Scott R. Manalis

Abstract Background: Mantle cell lymphoma (MCL) exhibits heterogeneous responses to targeted therapy, which are incompletely explained by genomic and immunophenotypic markers. Building on our prior work linking single-cell mass to BCR signaling activity and Bruton's tyrosine kinase inhibitors (BTKi) sensitivity, we hypothesized that baseline biophysical mass distributions may integrate proliferative, metabolic, and signaling states and predict clinical responses to BTKi-based therapy. Methods: Baseline tumor cells from treatment-naïve MCL patients in a phase 1/2 trial of Acalabrutinib, Venetoclax, and Obinutuzumab (NCT04855695) were enriched by fluorescence-activated cell sorting and analyzed via suspended microchannel resonators (SMR) without drug exposure to measure distributions of single-cell buoyant mass. Durable response (DR) was defined as achieving complete metabolic remission ≥12 months; non-durable response (NDR) as refractory disease or relapse within 12 months. Of the nine cases analyzed by SMR, seven also underwent parallel CyTOF profiling. Results: Baseline median mass of MCL cells was higher in NDR (n = 3; mean of patient medians 13.1 pg) than in DR (n = 6; mean of patient medians 13.1 pg; p = 0.02). The fraction of cells >15 pg—a threshold previously linked to BTK inhibitor response—was elevated in NDR (p=0.02) and correlated with clinical Ki-67 index (available in 6/9, r2=0.87). Eight of nine cases exhibited classic MCL morphology; the single blastoid case (NDR) had a median mass of 14.6 pg. In CyTOF-profiled samples, the >15 pg tumor cell fraction correlated with B-cell expression of Ki-67 (r2 = 0.99), the glucose transporter GLUT1 (r2 = 0.55), and the immune checkpoint ligand PD-L1 (r2 = 0.99), but not with the amino acid transporter CD98 or fatty acid transporter CD36. These associations are consistent with sequelae of heightened BCR. Across the cohort, an increased proportion of tumor cells >15 pg correlated with increased PD-1 expression on CD4+ T cells (r2 = 0.66) and expansion of T follicular helper (Tfh) cells (r2 = 0.74). Increased tumor cell mass distributions also positively correlated with Tfh activation markers, including ICOS, Ki-67, and checkpoint molecules TIGIT (r2 = 0.56-0.69). Taken together, these features raise the possibility of chronic B-cell antigen stimulation driving both Tfh activation and immune-regulatory feedback. Conclusions: Baseline MCL cell biophysical mass distributions correlate with tumor-intrinsic proliferation and metabolic activity as well as systemic Tfh activation and immune checkpoint expression. These results suggest that tumor biophysical properties ex vivo may capture in vivo BTKi sensitivity through tumor-intrinsic and immunologic mechanisms. Evaluation in larger cohorts and deeper mechanistic analyses are required to clarify its potential as a predictive biomarker. Citation Format: Mingzeng Zhang, Ye Zhang, Lydie Debaize, Grace Chen, Sona Baghiyan, Shogo Miura, Nezha Senhaji, Juniper Mai, Clare Phinney, Alexa Batingana, Jenalyn Weekes, Salah Abdulkarim, Haocheng Wang, Svitlana Tyekucheva, Jerome Ritz, Christine E. Ryan, Austin I. Kim, Scott R. Manalis, Mark A. Murakami. Baseline single-cell mass distributions correlate with clinical response to acalabrutinib, venetoclax, and obinutuzumab in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2442.

Cellular water content governs the concentration of all biomolecules inside a cell, thereby influencing the physical and functional properties of the cell. However, measurements of water content in physiologically relevant cell culture models remain largely unavailable, particularly in three-dimensional (3D) models such as tumor spheroids and organoids. Here, we achieve such measurements using an industrial-grade capillary steel tube. The steel tube functions as a mechanical resonator that inertially senses the buoyant mass of particles. For microgram-scale particles ≥ 400 micrometers in diameter, we achieve <1% precision error in buoyant mass with a 5-minute acquisition interval. By sequentially measuring the buoyant mass of individual, patient-derived glioblastoma tumor spheroids derived from patients with glioblastoma in media of different densities and cell permeabilities, we determine the absolute and fractional (volume/volume) water content of the spheroids, along with their dry mass, volume, and density properties. We achieve ~0.5% precision error in fractional water content with a throughput of three spheroids per hour. This enables us to detect both interspheroid heterogeneity in fractional water content and acute responses to kinase inhibition. Overall, we establish a simple and accessible technique for quantifying water content in living 3D cell culture models, opening previously unexplored avenues for studying biophysical regulation in multicellular systems.

ABSTRACT: Many cancer therapies induce high response rates, with some resulting in undetectable disease as assessed using standard clinical assays. This is particularly true in leukemia and lymphoma, in which patients often achieve deep remission yet ultimately experience relapse. These outcomes highlight a critical need to better understand ultrarare persistent cells that survive therapy but remain inaccessible to current techniques. Here, we developed Live-cell Pick-Seq (LiP-Seq), an advanced platform leveraging multiplexed live-cell imaging to identify and retrieve individual target cells for downstream analysis. LiP-Seq enables high-resolution transcriptomic profiling of single, viable lymphoma cells present at frequencies as low as 10-6, providing a technological window into the biology of these elusive reservoirs. Applying this method to patients with mantle cell lymphoma after treatment with CD19 chimeric antigen receptor T-cell (CAR-T) therapy, we identified recurrent upregulation of the immune modulator IFITM2 in the persistent cell fraction. Functional validation demonstrated that IFITM2 overexpression conferred protection against CAR-T cytotoxicity in vitro, implicating it as a potential survival mechanism under therapeutic pressure. Our results provide, to our knowledge, the first transcriptomic characterization of viable, ultrarare persistent cells using LiP-Seq, establishing a new paradigm for identifying and targeting features that may enable treatment evasion.

Cell growth rates exhibit cell-intrinsic cell-to-cell variability, which influences cell fitness and size homeostasis from bacteria to cancer. It remains unclear whether this variability arises from stochasticity in cell growth or division processes, or from cell-size-dependent growth regulation. To separate these potential sources of growth variability, single-cell growth rates need to be examined across different timescales. Here, we study cell size and growth regulation by tracking lymphocytic leukemia cell mass accumulation with high precision and minute-scale temporal resolution along long ancestral lineages. We first show that correlations between growth rates and cell-size nor asymmetric divisions explain cell-to-cell growth variability. We then isolate growth fluctuations by smoothing and detrending the growth rate dynamics using a Gaussian process regression. We find that these growth fluctuations drive cell-to-cell growth variability within ancestral lineages despite being independent of cell divisions, cell cycle, and cell size. Overall, our results provide a quantitative framework for understanding single-cell growth rates, and indicate that cell-intrinsic long-term patterns in growth are a byproduct of short-term growth fluctuations.

We present a new generation of Suspended Nanochannel Resonators (SNRs) featuring an optimized cantilever geometry enabling enhanced mass sensitivity and integrated within a micro-machined polymer cartridge acting as a fluidic interposer. This interposer provides backside fluidic access, mechanical clamping and straightforward coupling with external instrumentation. Compared to previous piezoresistive SNRs, the new devices incorporate thinner walls and higher quality factor. They were successfully applied to weigh individual 30 nm gold nanoparticles, achieving a 1.6 -fold improvement in mass sensitivity over previous designs of similar length.

Abstract Oxygen gradients organize tissue architecture and metabolism 1,2 , yet their precise spatial profiles and mechanistic roles remain poorly understood because both in vivo measurement and in vitro control are technically challenging 3,4 . Here, we quantify the oxygen landscape of the mammalian intestine using microscale sensors, revealing a steep luminal–basal gradient of approximately 10-60 µM mm − 1 that collapses under antibiotic perturbation. We then recreate this physiological range ex vivo with a submerged chemostat microfluidic platform that fixes the oxygen boundary condition by coupling an oxygen-permeable PDMS chip to an external scavenger reservoir and integrating embedded optical sensors for real-time readout. This architecture suppresses ambient oxygen ingress and sustains programmable gradients of 10-20 µM mm − 1 across three-dimensional colorectal cancer organoid cultures while remaining compatible with live imaging and endpoint retrieval. The platform bridges quantitative in vivo oxygen mapping with controlled ex vivo modeling, establishing a generalizable approach to interrogate how spatial oxygen dynamics govern epithelial organization and disease progression.

Intratumor heterogeneity impacts disease progression and therapeutic resistance but remains poorly characterized by conventional histologic, immunophenotypic, and molecular approaches. Single-cell biophysical properties distinguish functional phenotypes complementary to these approaches, providing additional insight into cellular diversity. Here, we link both buoyant mass and stiffness to gene expression to identify clinically relevant phenotypes within primary mantle cell lymphoma (MCL) cells, using MCL as a model of biological and clinical diversity in human cancer. Linked measurements reveal that buoyant mass and stiffness characterize B cell development states from naïve to plasma cell and correlate with expression of oncogenic B cell receptor signaling genes such as BLK and CD79A . In addition, changes in cell buoyant mass within primary patient specimens ex vivo correlate with sensitivity to Bruton’s tyrosine kinase inhibitors in vivo in MCL and chronic lymphocytic leukemia, another B cell malignancy. These findings highlight the value of biophysical properties as biomarkers of response in pursuit of future precision therapeutic strategies.

Marine phytoplankton face eco-evolutionary pressure to regulate their vertical position in the ocean to access light, which is abundant towards the surface, and nutrients, which are found deeper down the water column. All phytoplankton experience gravitational sinking, which can contribute to their vertical migration. However, the biophysical and molecular mechanisms that impact gravitational sinking have not been systematically characterized across taxa and environmental conditions. Here, we combine simulations with measurements of cell mass, volume, and composition to investigate the effects of nutrient availability on gravitational sinking in nine representative unicellular pico- and nanoplankton species. We find that gravitational sinking becomes faster in most species when starved, but the biophysical changes responsible for this vary across species and starvation conditions. For example, the faster sinking of Chaetoceros calcitrans is nearly exclusively driven by cell density whereas that of Emiliania huxleyi is due to cell volume. On the molecular level, the altered sinking is predominantly attributed to changes in cellular dry contents, rather than water. For example, starch accumulation increases sinking in three green algae species, and lipid accumulation decreases sinking in Phaeodactylum tricornutum. Overall, our work reveals that phytoplankton physiology has evolved multiple mechanisms that impact gravitational sinking in response to starvation, possibly to support the vertical migration of the cell.

Abstract Many cancer therapies induce high response rates, with some resulting in “undetectable” disease as assessed using standard pathology techniques, yet many patients ultimately relapse due to persistent minimal residual disease (MRD) and die of their disease. This is particularly true in patients with leukemia and lymphoma, including those receiving CD19-targeted chimeric antigen receptor (CAR) T-cell therapy for mantle cell lymphoma (MCL). Long-term follow-up of brexucabtagene autoleucel (KTE-X19) CAR T-cell therapy in R/R MCL reported 68% initial complete remissions (CRs), but only 37% progression-free survival at 3 years. Furthermore, only 60% of initially MRD-negative patients, assessed by next-generation sequencing (reported sensitivity: 1 in 105 cells), remained in extended remission. This highlights a critical need to better understand the MRD that persists after therapy. The limited ability of current techniques for characterizing ultra-rare cells has impaired the development of informed strategies to eradicate MRD. Although circulating tumor DNA detection enables MRD detection, capturing ultra-rare MRD cells to study phenotypes and vulnerabilities is challenging. Fluorescence-activated cell sorting and sequencing (Sort-Seq) can reliably isolate MRD cells at a sensitivity of only 10-4. To interrogate ultra-rare MRD, we developed Live-cell Pick-Seq (LiP-Seq), an advanced platform leveraging multiplexed live-cell imaging to identify and retrieve single tumor cells, enabling the isolation of viable lymphoma cells at extremely low frequency for single-cell RNA-sequencing (scRNA-seq). First, we validated LiP-Seq's capability to isolate cells at frequencies below 10−6 and compared its scRNA-seq quality against Sort-Seq in MCL patient samples. Among transcripts detected by both methods, relative expression correlated well between picked and sorted cells. Moreover, 98% of picked cells contained reads covering >= 5,000 genes (median 11,636 genes/cell), a higher fraction than obtained through sorting (88% covering >= 5000 genes, median 8,726 genes/cell). 5,262 genes were detected only in LiP-Seq but not Sort-Seq; these had lower expression than genes detected by both platforms. These data indicate that LiP-Seq generates high-quality scRNA-seq data and enables detection of lower-expressed genes. We then applied LiP-Seq to a clinical MRD setting. We collected 44 paired peripheral blood samples at multiple timepoints from 11 MCL patients before and in CR after CD19 CAR T-cell therapy (brexucabtagene autoleucel). Cells from these patients were picked based on an MCL-enriching panel (CD45+CD79b+CD5+CD3-) for scRNA-seq. To accurately identify tumor cells, we reconstructed BCR repertoires and developed a method to classify cells based on tumor-specific single nucleotide variants (SNVs). ScRNA-seq analysis of post-treatment MRD cells, particularly those identified as tumor-derived by SNV analysis, revealed upregulation of Interferon-Induced Transmembrane Protein 2 (IFITM2) compared to pre-treatment tumor cells. This upregulation was observed across multiple patients and timepoints. The IFITM family mediates cellular responses to interferons, and we have previously shown that IFITM3 acts as a scaffold to amplify PI3 kinase growth signaling in malignant and non-malignant B cells. More broadly, expression of IFITM1, IFITM2, or IFITM3 are predictors of poor prognosis in several cancers. We therefore assessed IFITM2 levels using in vitro co-culture assays of MCL with CD19-targeted CAR T cells. Strikingly, rare Jeko-1 cells that remained alive following 4 days of exposure to CD19-targeted CAR T cells expressed IFITM2 at 3-fold higher levels than cells cocultured with untransduced T cells. Moreover, exogenous overexpression of IFITM2 in Jeko-1 cells was sufficient to confer relative protection from CD19 CAR T-cell-mediated killing. These data, in combination with LiP-Seq of primary MRD specimens, highlight the cell surface protein IFITM2 as a potential mediator of MRD survival and resistance to CD19 CAR T-cell therapy in MCL. In summary, LiP-Seq offers a novel platform capable of isolating and characterizing viable ultra-rare target cells, providing unprecedented insights into MRD biology and facilitating downstream biological assays. In the setting of cancer MRD, this approach may identify important features with clinical relevance that are not obvious from studying more abundant tumor cells at other timepoints or disease states.

A bstract The growth of marine microbial communities drives biogeochemical cycling of carbon and other elements, yet the growth rates of individual species within complex ocean ecosystems remain poorly understood. In particular, the coexistence of a large diversity of copiotrophic bacteria, which are capable of fast growth but typically remain at low abundance, has been interpreted as a feast or famine existence. Here we show that contrary to the notion of infrequent growth, Vibrio bacteria exhibited consistent growth rates in coastal ocean samples, despite representing only a small fraction of the total community. These observations were enabled by a suspended microchannel resonator (SMR), which we adapted to function as a single-cell chemostat. By maintaining a continuous supply of native seawater around each trapped cell, we prevented nutrient depletion and used the SMR’s high mass precision to resolve growth rates that are otherwise undetectable. Vibrio species displayed significantly larger cell mass and faster growth than other community members across samples collected at different temporal intervals from days to years. Surprisingly, their growth was consistently limited by carbon, contrary to the expectation that heterotrophic bacteria in the euphotic zone would be limited by nitrogen and phosphorus due to competition with algae. The correlation between cell mass and growth rate of Vibrionaceae in seawater followed established growth laws derived from laboratory conditions, suggesting that growth physiology observed in pure cultures is applicable to wild bacterial populations. Overall, our findings suggest that rare species may play a disproportionately large role in the marine carbon cycle, with rapid biomass turnover driven by a combination of high growth rates balanced by intense predation.

Cellular water content governs the concentration of all biomolecules inside a cell, thereby influencing the physical and functional properties of the cell. However, measurements of water content in physiologically relevant cell culture models remain largely unavailable, particularly in 3D models such as tumor spheroids and organoids. Here, we achieve such measurements using a commercially available, industrial-grade, steel tube. The steel tube functions as a mechanical resonator that inertially senses the buoyant mass of particles. For microgram-scale particles ≥400 μm in diameter, we achieve <1% precision error in buoyant mass with a 5-minute acquisition interval. By sequentially measuring the buoyant mass of individual, glioblastoma patient-derived tumor spheroids in media of different densities and cell permeabilities, we determine the absolute and fractional (v/v) water content of the spheroids, along with their dry mass, volume, and density properties. We achieve ~0.4% precision error in fractional water content with a throughput of 3 spheroids per hour. This enables us to detect both inter-spheroid variability in fractional water content and acute responses to kinase inhibition. Overall, we establish a simple and accessible technique for quantifying water content in living 3D cell culture models, opening new avenues for studying biophysical regulation in multicellular systems.

Abstract Cell growth rates exhibit cell-intrinsic cell-to-cell variability, which influences cell fitness and size home-ostasis from bacteria to cancer. Whether this variability arises from noise in cell growth or cell division processes, or originates from cell-size-dependent growth rates, remains unclear. To separate these potential sources of growth variability, single-cell growth rates need to be examined across different timescales. Here, we study cell-intrinsic size and growth regulation by tracking lymphocytic leukemia cell mass accumulation with high precision and minute-scale temporal resolution along long ancestral lineages. We first show that cell-size-dependent growth regulation and asymmetric division of cell size do not explain cell-to-cell growth variability. We then isolate growth fluctuations from overlapping cell-cycle-dependent growth using a Gaussian process regression analysis. We find that these growth fluctuations drive cell-to-cell growth variability within ancestral lineages despite being independent of cell divisions, cell cycle, and cell size. Overall, our results indicate that cell-intrinsic long-term patterns in cell growth are a byproduct of short-term growth fluctuations.

Abstract Intra-tumor heterogeneity impacts disease progression and therapeutic resistance but remains poorly characterized by conventional histologic, immunophenotypic, and molecular approaches. Single-cell biophysical properties distinguish functional phenotypes complementary to these approaches, providing additional insight into cellular diversity. Here we link both buoyant mass and stiffness to gene expression to identify clinically relevant phenotypes within primary mantle cell lymphoma (MCL) cells, employing MCL as a model of biological and clinical diversity in human cancer. Linked measurements reveal that buoyant mass and stiffness characterize B-cell development states from naïve to plasma cell and correlate with expression of oncogenic B-cell receptor signaling genes such as BLK and CD79A . Additionally, changes in cell buoyant mass within primary patient specimens ex vivo correlate with sensitivity to Bruton’s Tyrosine Kinase inhibitors in vivo in MCL and chronic lymphocytic leukemia, another B-cell malignancy. These findings highlight the value of biophysical properties as biomarkers of response in pursuit of future precision therapeutic strategies.

Cancer cells alter their mechanical properties in response to the rigidity of their environment. Here, we explored the implications of this environmental mechanosensing for anti-tumor immunosurveillance using single cell biophysical profiling and metastasis models. Cancer cells stiffened in more rigid environments, a biophysical change that sensitized them to cytotoxic lymphocytes. In immunodeficient mice, this behavior manifested in the outgrowth of stiffer metastatic cells in the rigid bone than in the soft lung, while in immunocompetent hosts, it led to preferential elimination of stiffer cancer cells and suppression of bone metastasis. Environmentally-induced cell stiffening and immune sensitization both required Osteopontin, a secreted glycoprotein that is upregulated during bone colonization. Analysis of patient metastases spanning mechanically distinct tissues revealed associations between environmental rigidity, immune infiltration, and cancer cell stiffness consistent with mechanically driven immunosurveillance. These results demonstrate how environmental mechanosensing modulates anti-tumor immunity and suggest a mechanoimmunological basis for metastatic site selection.

ABSTRACT Marine phytoplankton face eco-evolutionary pressure to regulate their vertical position in the ocean to access light, which is abundant towards the surface, and nutrients, which are found deeper down the water column. All phytoplankton experience gravitational sinking, which can contribute to their vertical migration. However, the biophysical and molecular mechanisms that impact gravitational sinking have not been systematically characterized across taxa and environmental conditions. Here, we combine simulations with measurements of cell mass, volume, and composition to investigate the effects of nutrient availability on gravitational sinking in 9 representative unicellular pico- and nanoplankton species. We find that gravitational sinking becomes faster in most species when starved, but the biophysical changes responsible for this vary across species and starvation conditions. For example, the faster sinking of Chaetoceros calcitrans is nearly exclusively driven by cell density whereas that of Emiliania huxleyi is due to cell volume. On the molecular level, the altered sinking is predominantly attributed to changes in cellular dry contents, rather than water. For example, starch accumulation increases sinking in 3 green algae species, and lipid accumulation decreases sinking in Phaeodactylum tricornutum . Overall, our work reveals that phytoplankton physiology has evolved multiple mechanisms that impact gravitational sinking in response to starvation, possibly to support the vertical migration of the cell.

Abstract Advancements in measuring the biophysical properties of single cells with both high precision and throughput have furthered our understanding of biological processes and facilitated the development of innovative clinical biomarkers. These biomarkers offer a comprehensive assessment of cell state with utility for both research and clinical contexts. This presentation will: (i) demonstrate the capability of single-cell mass measurements for guiding therapy in cancer treatment; and (ii) present a novel approach to quantify the fractional water content of single cells which serves as a proxy for molecular crowding. Our findings suggest that, unlike mass, proliferating cells maintain strict regulation over their water content. However, biological processes or perturbations that impact proliferation can lead to dramatic changes in water content. Thus, water content emerges as a crucial indicator of cell state, with potential implications for disease diagnosis and monitoring. Citation Format: Scott Manalis. Measuring single-cell mass: Biological insights and clinical translation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr IA020.

Background and Significance: Measurable residual disease (MRD) represents the fundamental driver of relapse and mortality in acute myeloid leukemia (AML). However, there are currently no established approaches to address this unmet need. Our central hypothesis is that targeting vulnerabilities associated with specific leukemia genotypes will eradicate MRD and prevent disease relapse. Break Through Cancer is a collaboration between our centers aimed at making progress in the deadliest cancers by stimulating radical clinical and laboratory research collaboration. The menin-KMT2A interaction is a critical dependency in acute leukemias caused by rearrangement of the Lysine methyl transferase (KMT2Ar) or Nucleoporin 98 (NUP98r) genes, or mutation of the Nucleophosmin 1 gene (NPM1mt). Revumenib (previously SNDX-5613), is a potent, oral, selective inhibitor of this interaction with an established safety and efficacy in refractory leukemias with these genotypes (Issa GC, Nature 2023). Additionally, KMT2Ar or NPM1mt leukemias can undergo BCL2-dependent apoptosis, and dual Bcl-2 and menin inhibition led to synergistic activity in KMT2Ar or NPM1mt leukemia models (Carter BZ, Blood 2021; Fiskus W, BCJ 2022). Therefore, we designed this study investigating the combination of revumenib and venetoclax to eradicate MRD in these AML subtypes (NCT06284486). Study Design and Methods: This is a single arm, open label, multicenter, phase I/II investigator-initiated study. Patients (pts) age ≥ 12 years with weight ≥ 45Kg, and known history of NPM1mt, or KMT2Ar, or NUP98r AML with MRD ≥ 0.1% identified by multiparameter flow cytometry (MFC) using central testing would be eligible; no morphologic evidence of AML (blasts &amp;lt;5%) in first remission following high intensity therapy or at least 2 cycles of low intensity therapy, or in second remission following any therapy. Up to 12 pts will be enrolled on the phase I, using 3+3, with escalating doses of revumenib and a target dose of 163 mg PO Q12h (with strong CYP3A inhibitor) or 276 mg PO Q12h (without strong CYP3A inhibitor) days 1-28, with venetoclax 400 mg (target dose) PO daily, days 1-14. The primary objective of the phase I is to determine safety, and the recommended phase II dose. The primary objective of the phase II is to assess the efficacy of venetoclax and revumenib in clearance of MRD (conversion to undetectable by central MFC) within 8 cycles. Up to 14 pts will be included on the phase II portion of the study. With a sample size of 20 pts (Phase II + RP2D in Phase I), the power is 76% assuming an MRD clearance of 30% (based on QUAZAR trial, Roboz et al. Blood 2022) against a null rate of 8%, by a two-sided Fisher's exact test at a significance level of 0.05. We plan to monitor futility and toxicity where enrollment will be stopped early if &amp;gt;95% probability that the MRD conversion rate is &amp;lt; 30% or there is &amp;gt;90% probability that the unacceptable toxicity rate &amp;gt;20%. Secondary objectives include assessment of duration of response, event-free and overall survival and concordance of genetic and flow MRD. This trial includes longitudinal collection of samples, with exploratory objectives focused on improving MRD detection using cell-free DNA, single-cell mutation analysis and cytometry by time of flight. In addition, we aim to use various models in addition to patient samples to improve understanding of MRD biology and identify novel susceptibilities. Accrual is planned at MD Anderson, Dana Farber, Memorial Sloan Kettering, and Johns Hopkins. This study may identify a novel strategy to eradicate MRD using combination targeted therapies which may decrease recurrence and improve remission duration. In addition, this study could improve detection of MRD, and our understanding of MRD biology.

ABSTRACT Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling and are promising bioproduction chassis. Here, we isolate two novel cyanobacteria with 4.6Mbp genomes, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on a solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (&gt;31 g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting favorable economics of harvesting UTEX 3222 biomass. These traits may make UTEX 3222 a compelling choice for marine carbon dioxide removal (CDR) and photosynthetic bioproduction from CO₂. Overall, we find that bio-prospecting in environments with naturally elevated CO₂ may uncover novel CO₂-metabolizing organisms with unique characteristics. IMPORTANCE Cyanobacteria provide a potential avenue for both biomanufacturing and combatting climate change via high-efficiency photosynthetic carbon sequestration. This study identifies novel photosynthetic organisms isolated from a unique geochemical environment and describes their genomes, growth behavior in culture, and biochemical composition. These cyanobacteria appear to make a tractable research model, and cultures are made publicly available alongside information about their culture and maintenance. Application of these organisms to carbon sequestration and/or biomanufacturing is discussed, including unusual, rapid settling characteristics of the strains relevant to scaled culture.

Current processes for the production of recombinant adeno-associated virus (rAAV) are inadequate to meet the surging demand for rAAV-based gene therapies. This article reviews recent advances that hold the potential to address current limitations in rAAV manufacturing. A multidisciplinary perspective on technological progress in rAAV production is presented, underscoring the necessity to move beyond incremental refinements and adopt a holistic strategy to address existing challenges. Since several recent reviews have thoroughly covered advancements in upstream technology, this article provides only a concise overview of these developments before moving to pivotal areas of rAAV manufacturing not well covered in other reviews, including analytical technologies for rapid and high-throughput measurement of rAAV quality attributes, mathematical modeling for platform and process optimization, and downstream approaches to maximize efficiency and rAAV yield. Novel technologies that have the potential to address the current gaps in rAAV manufacturing are highlighted. Implementation challenges and future research directions are critically discussed.

Eukaryotic phytoplankton, also known as algae, form the basis of marine food webs and drive marine carbon sequestration. Algae must regulate their motility and gravitational sinking to balance access to light at the surface and nutrients in deeper layers. However, the regulation of gravitational sinking remains largely unknown, especially in motile species. Here, we quantify gravitational sinking velocities according to Stokes’ law in diverse clades of unicellular marine microalgae to reveal the cell size, density, and nutrient dependency of sinking velocities. We identify a motile algal species, Tetraselmis sp., that sinks faster when starved due to a photosynthesis-driven accumulation of carbohydrates and a loss of intracellular water, both of which increase cell density. Moreover, the regulation of cell sinking velocities is connected to proliferation and can respond to multiple nutrients. Overall, our work elucidates how cell size and density respond to environmental conditions to drive the vertical migration of motile algae.

All cells are subject to geometric constraints, including the surface area-to-volume (SA/V) ratio, which can limit nutrient uptake, maximum cell size, and cell shape changes. Like the SA/V ratio of a sphere, it is generally assumed that the SA/V ratio of cells decreases as cell size increases. However, the structural complexity of the plasma membrane makes studies of the surface area challenging in cells that lack a cell wall. Here, we investigate near-spherical mammalian cells using single-cell measurements of cell mass and plasma membrane proteins and lipids, which allows us to examine the cell size scaling of cell surface components as a proxy for the SA/V ratio. Surprisingly, in various proliferating cell lines, cell surface components scale proportionally with cell size, indicating a nearly constant SA/V ratio as cells grow larger. This behavior is largely independent of the cell cycle stage and is also observed in quiescent cells, including primary human monocytes. Moreover, the constant SA/V ratio persists when cell size increases excessively during polyploidization. This is enabled by increased plasma membrane folding in larger cells, as verified by electron microscopy. We also observe that specific cell surface proteins and cholesterol can deviate from the proportional size scaling. Overall, maintaining a constant SA/V ratio ensures sufficient plasma membrane area for critical functions such as cell division, nutrient uptake, growth, and deformation across a wide range of cell sizes.

SUMMARY Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples. HIGHLIGHTS Relapse after remission on TKI can harbor mutations in ABL1, RAS, or neither Mutations and development-like cell state dictate fitness in residual disease Co-targeting cell state and ABL1 markedly reduces MRD Biophysical measurements provide an integrative, rapid measurement of cell state

Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and therefore a fundamental determinant of cell state and function. However, existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples. Here we present an approach for measuring the density of 30,000 single cells per hour with a precision of 0.03% (0.0003 g/mL) by integrating fluorescence exclusion microscopy with a suspended microchannel resonator. Applying this approach to human lymphocytes, we discovered that cell density and its variation decrease as cells transition from quiescence to a proliferative state, suggesting that the level of molecular crowding decreases and becomes more regulated upon entry into the cell cycle. Using a pancreatic cancer patient-derived xenograft model, we found that the ex vivo density response of primary tumor cells to drug treatment can predict in vivo tumor growth response. Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a new biomarker for functional precision medicine.

Leukemias and their bone marrow microenvironments undergo dynamic changes over the course of disease. However, little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of CLC dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1-2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: (i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and (ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells.

for vitrification. To benchmark our platform with the manual method, we vitrified over a hundred mouse eggs and found comparable percentages (∼95%) for post-vitrification survivability. In addition, our platform performs real-time microscopy of the egg thereby enabling future studies where its morphology may be linked to functional outcomes. Our study contributes to the ongoing efforts to enhance the automation of embryology techniques towards broader applications in reproductive medicine both for clinical and research purposes.

Abstract Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling as well as promising bioproduction chassis. Here, we isolate two novel cyanobacteria, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically-relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (&gt;31g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting improved de-watering of UTEX 3222 biomass. This settling behavior can be explained in part by larger cell volume. These traits may make UTEX 3222 a compelling choice for photosynthetic bioproduction from CO₂. Overall, we find that bio-prospecting in environments with naturally elevated CO₂ may uncover novel CO₂-metabolizing organisms with unique characteristics.

ABSTRACT Optical density is a proxy of total biomass concentration and is commonly used for measuring the growth of bacterial cultures. However, there is a misconception that exponential optical density growth is equivalent to steady-state population growth. Many cells comprise a culture and individuals can differ from one another. Hallmarks of steady-state population growth are stable frequency distributions of cellular properties over time, something total biomass growth alone cannot quantify. Using single-cell mass sensors paired with optical density measurements, we explore when steady-state population growth prevails in typical batch cultures. We find the average cell mass of Escherichia coli and Vibrio cyclitrophicus growing in several media increases by 0.5–1 orders of magnitude within a few hours of inoculation, and that time-invariant mass distributions are only achieved for short periods when cultures are inoculated with low initial biomass concentrations from overnight cultures. These species achieve an effective steady-state after approximately 2.5–4 total biomass doublings in rich media, which can be decomposed to 1 doubling of cell number and 1.5–3 doublings of average cell mass. We also show that typical batch cultures in rich media depart steady-state early in their growth curves at low cell and biomass concentrations. Achieving steady-state population growth in batch culture is a delicate balancing act, so we provide general guidance for commonly used rich media. Quantifying single-cell mass outside of steady-state population growth is an important first step toward understanding how microbes grow in their natural context, where fluctuations pervade at the scale of individuals. IMPORTANCE Microbiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates from optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells. Here, we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for individual cell mass accumulation and cell number increase via cell division to balance out. Steady-state growth is important for reliable cell mass distributions and experimental reproducibility. We discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.

Leukemias and their bone marrow microenvironment are known to undergo dynamic changes over the course of disease. However, relatively little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of leukemia cell dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1-2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells.

Topic: 20. Lymphoma Biology & Translational Research Background: Most patients with mantle cell lymphoma (MCL) achieve complete remission with front line therapy, but relapses invariably occur due to the persistence, evolution, and expansion of drug-tolerant lymphoma cell subpopulations. This biologic heterogeneity has hindered efforts to identify tolerable precision therapies capable of overcoming residual disease and curing patients with MCL. Aims: We aim to resolve biological heterogeneity in MCL with single-cell resolution by integrating functional biophysical properties (mass and stiffness) with transcriptomic and genetic features. Specifically, we seek to define ex vivo biomarkers of clinical response or resistance in primary biospecimens from patients with MCL treated on an ongoing phase I/II trial of acalabrutinib, venetoclax, and obinutuzumab (AVO). Methods: We acquired synchronous blood, bone marrow and lymph node biopsies from patients with MCL treated with AVO on clinical trial NCT04855695 prior to treatment, followed when available by blood and bone marrow at clinical remission and post-progression. Biophysical properties of individual cells were measured using suspended microchannel resonators (SMR) in serial with single-cell transcriptomics via Seq-Well. Somatic copy-number variants (SCNVs) were inferred from transcriptome read counts. Additionally, we linked biophysical measurements of individual MCL cells from three patient-derived xenograft (PDX) models of MCL to single-cell transcriptomes via Smart-Seq2 (scSMR/RNA-seq). Results: Within the first four enrolled patients, we observed inter- and intra-tumor heterogeneity in mass and stiffness. In parallel, 33,171 single cells were sequenced across 17 samples spanning the pre-treatment to progression continuum. Notably, one patient with blastoid/pleomorphic histology and a TP53 mutation progressed on AVO treatment. Serial analyses demonstrated biophysical correlates of histologic transformation, with a significant shift toward higher single-cell mass distributions in blastoid/pleomorphic cells. Linked single-cell transcriptomes suggested the acquisition of new SCNVs in the malignant cells at progression. These cells were also distinguished by increased mTORC1 and oxidative phosphorylation signaling, as previously described in BTK-inhibitor resistance, but independently of TLR/CD40-mediated activation, suggesting potential metabolic rewiring. Finally, tumor cells at progression were strongly associated with an active cell proliferation state and enriched in genes involved in ATM signaling, DNA damage response and B cell receptor (BCR) signaling pathway, suggesting aberrant activation of BTK-mediated proliferation while on acalabrutinib therapy. These results corroborate prior data from in vitro activation assays and preclinical models linking biophysical phenotypes with B cell transcriptional states, including developmental stage, histological subtype, cell cycle and BCR activity. Here, application of scSMR/RNA-seq to three PDXs models of MCL revealed that mass and stiffness correlate with genes annotated for cell division, cell cycle, and BCR signaling pathway. Summary/Conclusion: The combination of serial orthogonal biophysical and multi-omic molecular profiling at single-cell resolution can nominate potential mechanisms of drug resistance. Our long-term goal is to leverage biophysical properties to resolve functionally resistant cellular subclones in which to focus single-cell multi-omics to nominate candidate targets for precision therapeutic strategies. Keywords: Clinical trial, Bruton’s tyrosine kinase inhibitor (BTKi), Mantle cell lymphoma, Drug resistance

ABSTRACT Eukaryotic phytoplankton, also known as algae, form the basis of marine food webs and drive marine carbon sequestration when their biomass sinks to the ocean floor. Algae must regulate their vertical movement, as determined by motility and gravitational sinking, to balance access to light at the surface and nutrients in deeper layers. However, the regulation of gravitational sinking velocities remains largely unknown, especially in motile species. Here, we directly quantify single-cell masses and volumes to calculate sinking velocities according to Stokes’ law in diverse clades of unicellular marine microalgae. Our results reveal the cell size, light, and nutrient-dependency of sinking velocities. We identify motile dinoflagellate and green algal species that increase their sinking velocity in response to starvation. Mechanistically, this increased cell sinking is achieved by photosynthesis-driven accumulation of carbohydrates, which increases cell mass and density. Moreover, cell sinking velocities correlate inversely with proliferation rates, and the mechanism regulating cell sinking velocities integrates signals from multiple nutrients. Our findings suggest that the regulation of cell composition according to environmental conditions contributes to the vertical movement of motile cells in the oceans. More broadly, our approach for sinking velocity measurements expands the study of gravitational sinking to motile cells and supports the modeling of marine carbon pump and nutrient cycles.

&lt;p&gt;Supplementary Table 1, Table 2 and Table 3&lt;/p&gt;

&lt;p&gt;Supplementary Figures&lt;/p&gt;

&lt;p&gt;Supplementary Figures&lt;/p&gt;

&lt;p&gt;Supplementary Table 1, Table 2 and Table 3&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we described the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane–bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors.&lt;/p&gt;Significance:&lt;p&gt;The oncogenic form of HSP90 organizes and maintains functional multienzymatic metabolic hubs in cancer cells, suggesting the potential of repurposing oncogenic HSP90 selective inhibitors to disrupt metabolism in lymphoma cells.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we described the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane–bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors.&lt;/p&gt;Significance:&lt;p&gt;The oncogenic form of HSP90 organizes and maintains functional multienzymatic metabolic hubs in cancer cells, suggesting the potential of repurposing oncogenic HSP90 selective inhibitors to disrupt metabolism in lymphoma cells.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Movie over 12 hours of the head of a zebrafish embryo injected with EV control A375 cells.&lt;/p&gt;

&lt;p&gt;YAP-AA does not enhance entry into circulation from the injection site by 10HPI.&lt;/p&gt;