An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Mechanisms and therapeutic implications of hypermutation in gliomas.

A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

Hypercholesterolemia induces angiogenesis and accelerates growth of breast tumors in vivo.

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

A pilot study to investigate the safety and feasibility of antiretroviral therapy for Alzheimer's disease (ART-AD).

Retrotransposons are viral-like DNA sequences that constitute approximately 41% of the human genome. Studies in Drosophila, mice, cultured cells, and human brain indicate that retrotransposons are activated in settings of tauopathy, including Alzheimer's disease, and causally drive neurodegeneration. The anti-retroviral medication 3TC (lamivudine), a nucleoside analog reverse transcriptase inhibitor, limits retrotransposon activation and suppresses neurodegeneration in tau transgenic Drosophila, two mouse models of tauopathy, and in brain assembloids derived from patients with sporadic Alzheimer's disease. We performed a 24-week phase 2a open-label clinical trial of 300 mg daily oral 3TC (NCT04552795) in 12 participants aged 52-83 years with a diagnosis of mild cognitive impairment due to suspected Alzheimer's disease. Primary outcomes included feasibility, blood brain barrier penetration, effects of 3TC on reverse transcriptase activity in the periphery, and safety. Secondary outcomes included changes in cognition and fluid-based biomarkers of neurodegeneration and neuroinflammation. All participants completed the six-month trial; one event of gastrointestinal bleeding due to a peptic ulcer was reported. 3TC was detected in blood and cerebrospinal fluid (CSF) of all participants, suggestive of adherence to study drug and effective brain penetration. Cognitive measures remained stable throughout the study. Glial fibrillary acidic protein (GFAP) (P=0.03) and Flt1 (P=0.05) were significantly reduced in CSF over the treatment period; Aß42/40 (P=0.009) and IL-15 (P=0.006) were significantly elevated in plasma. While this is an open label study of small sample size, the significant decrease of some neurodegeneration- and neuroinflammation-related biomarkers in CSF, significantly elevated levels of plasma Aß42/40, and a trending decrease of CSF NfL after six months of 3TC exposure suggest a beneficial effect on subjects with mild cognitive impairment due to suspected Alzheimer's disease. Feasibility, safety, tolerability, and central nervous system (CNS) penetration assessments further support clinical evaluation of 3TC in a larger placebo-controlled, multi-dose clinical trial.

Hypercholesterolemia promotes an osteoporotic phenotype.

A role for hypercholesterolemia in the development of osteoporosis has been suggested in published reports. However, few studies contain direct evidence of a role for maintenance of cholesterol homeostasis in bone health. Using isocaloric high-fat/high-cholesterol and low-fat/no-cholesterol diets in a 4-month feeding study combined with micro computed tomography analysis, we demonstrated in two different mouse strains that mice with hypercholesterolemia lose cortical and trabecular bone in the femurs and vertebrae (bone mineral density was decreased on average by ≈90 mg/mL in the cortical vertebrae in one strain) and cortical bone in the calvariae (bone mineral density was decreased on average by ≈60 mg/mL in one strain). Mechanical testing of the femurs demonstrated that loss of bone in the mice with hypercholesterolemia caused changes in the mechanical properties of the bone including loss of failure load (failure load was decreased by ≈10 N in one strain) and energy to failure. Serologic and histomorphologic analyses suggested that hypercholesterolemia promotes osteoclastogenesis. These studies support a role for hypercholesterolemia in the development of osteoporosis and provide a model with which to test intervention strategies to reduce the effects of hypercholesterolemia on bone health.

Increased smooth muscle contractility in mice deficient for neuropilin 2.

Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2(+/LacZ) and Nrp2(+/gfp) transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2, compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility.

ALK Amplification and Rearrangements Are Recurrent Targetable Events in Congenital and Adult Glioblastoma.

PURPOSE: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. RESULTS: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. CONCLUSIONS: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. See related commentary by Mack and Bertrand, p. 2567.

FosB regulates stretch-induced expression of extracellular matrix proteins in smooth muscle.

Fibroproliferative remodeling in smooth muscle-rich hollow organs is associated with aberrant extracellular matrix (ECM) production. Although mechanical stimuli regulate ECM protein expression, the transcriptional mediators of this process remain poorly defined. Previously, we implicated AP-1 as a mediator of smooth muscle cell (SMC) mechanotransduction; however, its role in stretch-induced ECM regulation has not been explored. Herein, we identify a novel role for the AP-1 subunit FosB in stretch-induced ECM expression in SMCs. The DNA-binding activity of AP-1 increased after stretch stimulation of SMCs in vitro. In contrast to c-Jun and c-fos, which are also activated by the SMC mitogen platelet-derived growth factor, FosB was only activated by stretch. FosB silencing attenuated the expression of the profibrotic factors tenascin C (TNC) and connective tissue growth factor (CTGF), whereas forced expression of Jun~FosB stimulated TNC and CTGF promoter activity. Chromatin immunoprecipitation revealed enrichment of AP-1 at the TNC and CTGF promoters. Bladder distension in vivo enhanced nuclear localization of c-jun and FosB. Finally, the distension-induced expression of TNC and CTGF in the detrusor smooth muscle of bladders from wild-type mice was significantly attenuated in FosB-null mice. Together, these findings identify FosB as a mechanosensitive regulator of ECM production in smooth muscle.

Synthetic extracellular matrices and astrocytes provide a supportive microenvironment for the cultivation and investigation of primary pediatric gliomas.

Background: Pediatric gliomas comprise a diverse set of brain tumor entities that have substantial long-term ramifications for patient survival and quality of life. However, the study of these tumors is currently limited due to a lack of authentic models. Additionally, many aspects of pediatric brain tumor biology, such as tumor cell invasiveness, have been difficult to study with currently available tools. To address these issues, we developed a synthetic extracellular matrix (sECM)-based culture system to grow and study primary pediatric brain tumor cells. Methods: We developed a brain-like sECM material as a supportive scaffold for the culture of primary, patient-derived pediatric glioma cells and established patient-derived cell lines. Primary juvenile brainstem-derived murine astrocytes were used as a feeder layer to support the growth of primary human tumor cells. Results: We found that our culture system facilitated the proliferation of various primary pediatric brain tumors, including low-grade gliomas, and enabled ex vivo testing of investigational therapeutics. Additionally, we found that tuning this sECM material allowed us to assess high-grade pediatric glioma cell invasion and evaluate therapeutic interventions targeting invasive behavior. Conclusion: Our sECM culture platform provides a multipurpose tool for pediatric brain tumor researchers that enables both a wide breadth of biological assays and the cultivation of diverse tumor types.

FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits.

Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. SIGNIFICANCE: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977.

PPM1D mutations are oncogenic drivers of de novo diffuse midline glioma formation.

The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.

Prohibitin is a cholesterol-sensitive regulator of cell cycle transit.

Cholesterol is essential in establishing most functional animal cell membranes; cells cannot grow or proliferate in the absence of sufficient cholesterol. Consequently, almost every cell, tissue, and animal tightly regulates cholesterol homeostasis, including complex mechanisms of synthesis, transport, uptake, and disposition of cholesterol molecules. We hypothesize that cellular recognition of cholesterol insufficiency causes cell cycle arrest in order to avoid a catastrophic failure in membrane synthesis. Here, we demonstrate using unbiased proteomics and standard biochemistry that cholesterol insufficiency causes upregulation of prohibitin, an inhibitor of cell cycle progression, through activation of a cholesterol-responsive promoter element. We also demonstrate that prohibitin protects cells from apoptosis caused by cholesterol insufficiency. This is the first study tying cholesterol homeostasis to a specific cell cycle regulator that inhibits apoptosis.

Ezetimibe is an inhibitor of tumor angiogenesis.

Epidemiological and preclinical observations have suggested a role for one or more products of the mevalonate/cholesterol biosynthesis pathway in the progression of prostate cancer. In this study, we used ezetimibe (Zetia), a specific, FDA-approved, cholesterol uptake-blocking drug, in combination with either a hyper- or hypocholesterolemic diet, to show that elevated circulating cholesterol levels promote, whereas a reduction in circulating cholesterol levels retard, the growth of human prostate cancer xenograft tumors in mice. Circulating cholesterol levels also modified tumor angiogenesis; higher cholesterol levels increased microvessel density and other indicators of vascularity. Consistent with these data, the reduction of cholesterol levels also increased the levels of the angiogenesis inhibitor thrombospondin-1 in the xenografts. Our results thus suggest that hypercholesterolemia directly accelerates the growth of prostate carcinomas, and that the pharmacological reduction of serum cholesterol levels may retard prostate cancer growth by inhibiting tumor angiogenesis.

Ezetimibe reduces enlarged prostate in an animal model of benign prostatic hyperplasia.

PURPOSE: Benign prostatic hyperplasia is a common urinary tract disorder that affects aging men. The molecular mechanisms underlying benign prostatic hyperplasia are obscure and the development of animal models to test novel treatment strategies is challenging. We report that the Bio 87.20 hamster strain (Bio Breeders, Watertown, Massachusetts) shows 5α-reductase-sensitive prostate enlargement and a decrease in circulating cholesterol reduces prostate size. MATERIALS AND METHODS: Bio 87.20 hamsters 17 months old with an enlarged prostate were fed a diet containing no or minimal cholesterol and including finasteride (Merck, Whitehouse Station, New Jersey) and/or ezetimibe (Schering-Plough, Kenilworth, New Jersey) for 4 months. The prostate complex was removed, volume and weight were determined, and tissue was examined histologically. RESULTS: Prostate enlargement depended on cholesterol in the diet. Blockade of intestinal cholesterol transport with ezetimibe induced prostate regression to a similar extent as the 5α-reductase inhibitor finasteride, a compound used to treat benign prostatic hyperplasia in humans. Histological analysis indicated that finasteride induced widespread prostatic atrophy but normal glandular architecture was preserved in the ezetimibe cohort. CONCLUSIONS: Results indicate that dysregulation of cholesterol metabolism may be a component of benign prostatic hyperplasia and ezetimibe may be effective as an alternative or adjunct to standard treatment. Our findings also show that the Bio 87.20 hamster is a suitable model for preclinical evaluation of novel benign prostatic hyperplasia therapy.

Single-Cell RNA-Seq Reveals Cellular Hierarchies and Impaired Developmental Trajectories in Pediatric Ependymoma.

Ependymoma is a heterogeneous entity of central nervous system tumors with well-established molecular groups. Here, we apply single-cell RNA sequencing to analyze ependymomas across molecular groups and anatomic locations to investigate their intratumoral heterogeneity and developmental origins. Ependymomas are composed of a cellular hierarchy initiating from undifferentiated populations, which undergo impaired differentiation toward three lineages of neuronal-glial fate specification. While prognostically favorable groups of ependymoma predominantly harbor differentiated cells, aggressive groups are enriched for undifferentiated cell populations. The delineated transcriptomic signatures correlate with patient survival and define molecular dependencies for targeted treatment approaches. Taken together, our analyses reveal a developmental hierarchy underlying ependymomas relevant to biological and clinical behavior.

Mitogenic and progenitor gene programmes in single pilocytic astrocytoma cells.

Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.

Buparlisib in Patients With Recurrent Glioblastoma Harboring Phosphatidylinositol 3-Kinase Pathway Activation: An Open-Label, Multicenter, Multi-Arm, Phase II Trial.

PURPOSE: Phosphatidylinositol 3-kinase (PI3K) signaling is highly active in glioblastomas. We assessed pharmacokinetics, pharmacodynamics, and efficacy of the pan-PI3K inhibitor buparlisib in patients with recurrent glioblastoma with PI3K pathway activation. METHODS: This study was a multicenter, open-label, multi-arm, phase II trial in patients with PI3K pathway-activated glioblastoma at first or second recurrence. In cohort 1, patients scheduled for re-operation after progression received buparlisib for 7 to 13 days before surgery to evaluate brain penetration and modulation of the PI3K pathway in resected tumor tissue. In cohort 2, patients not eligible for re-operation received buparlisib until progression or unacceptable toxicity. Once daily oral buparlisib 100 mg was administered on a continuous 28-day schedule. Primary end points were PI3K pathway inhibition in tumor tissue and buparlisib pharmacokinetics in cohort 1 and 6-month progression-free survival (PFS6) in cohort 2. RESULTS: Sixty-five patients were treated (cohort 1, n = 15; cohort 2, n = 50). In cohort 1, reduction of phosphorylated AKTS473 immunohistochemistry score was achieved in six (42.8%) of 14 patients, but effects on phosphoribosomal protein S6S235/236 and proliferation were not significant. Tumor-to-plasma drug level was 1.0. In cohort 2, four (8%) of 50 patients reached 6-month PFS6, and the median PFS was 1.7 months (95% CI, 1.4 to 1.8 months). The most common grade 3 or greater adverse events related to treatment were lipase elevation (n = 7 [10.8%]), fatigue (n = 4 [6.2%]), hyperglycemia (n = 3 [4.6%]), and elevated ALT (n = 3 [4.6%]). CONCLUSION: Buparlisib had minimal single-agent efficacy in patients with PI3K-activated recurrent glioblastoma. Although buparlisib achieved significant brain penetration, the lack of clinical efficacy was explained by incomplete blockade of the PI3K pathway in tumor tissue. Integrative results suggest that additional study of PI3K inhibitors that achieve more-complete pathway inhibition may still be warranted.

Linking single-cell measurements of mass, growth rate, and gene expression.

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.