Prostate cancer cell invasion into the surrounding muscle capsule, known as extracapsular extension (ECE), is associated with a significantly worse prognosis (5-year survival reduction: 98% to 31%). Using a live ECE mouse xenograft model with DU145 tumor cells, our group previously demonstrated that functional heterogeneity among adhesion subtypes within invasive clusters is essential for muscle invasion. Bulk RNA sequencing from three spatial compartments (pre-invasive, invasive, and post-invasive) revealed distinct gene expression profiles, with hypoxia-related and cell–cell/cell–ECM interaction genes prominently dysregulated in muscle-invasive disease. In this study, we sought to define the single-cell transcriptional heterogeneity across the three ECE compartments and compare these patterns to a human patient-derived xenograft (PDX) model. Spatial transcriptomic analysis (STA) was performed using the Bruker CosMx platform with a 6K gene panel. Data was preprocessed (quality control and dimensional reduction) and analyzed in R 4.4.0 using Seurat v5. Notable downstream analyses included unsupervised Louvain clustering, trajectory inference with Monocle 3, and cluster-specific differential gene expression. In the ECE model, STA resolved at least 11 distinct tumor cell identity clusters previously undetectable by bulk methods and revealed a clear spatial demarcation between pre-invasive and invasive populations crossing the muscle barrier. Trajectory analysis identified a pre-invasive population branching into two invasive fates with unique expression profiles. Dispersion scores, calculated using principal component embeddings, exhibited progressively increasing intracluster heterogeneity along one branch. Strong expression enrichment of the Prolaris 31-gene prognostic panel, a clinically established marker of aggressiveness, was observed at the bifurcation point of tumor evolution. STA of the human PDX model similarly revealed a high-dispersion invasive cluster branching into two fates, one exhibiting decreased heterogeneity consistent with specialization of invasive phenotypes. Together, these findings reveal, for the first time, that tumor invasion of the extracapsular muscle layer induces substantial transcriptional heterogeneity. This dynamic process highlights potential therapeutic vulnerabilities during the transition to extracapsular extension. Targeting tumor cells at this critical juncture may represent a key strategy for preventing ECE and improving prostate cancer outcomes. Supported by P30CA023074, University of Arizona Comprehensive Cancer Center Support Grant. Saptarshi Mallick, Rafael Sainz, Jayati Chakrabarti, Kendra D. Marr, Noel A. Warfel, Gregory C. Rogers, Beatrice S. Knudsen, Rashid Sayyid, Kelvin W. Pond, Anne E. Cress. Single cell spatial transcriptomics identifies unique cell populations driving muscle invasive prostate adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B045.

Abstract Aggressive human prostate tumors traverse a contractile smooth muscle pseudo-capsule, in a process termed extracapsular extension (ECE), defining the pT3 pathologic stage associating with increased biochemical recurrence, bone metastases, and cancer-specific mortality. While T3 lesions can be detected by non-invasive mpMRI imaging, how the tumor invades into and through the contractile muscle is unknown. Using a mouse xenograft model system of ECE, we investigated the transcriptomic consequences of human tumor invasion into and through the contractile smooth muscle of the mouse diaphragm. Both human and mouse gene transcripts were documented and analyzed. Tumor cells were intraperitoneally injected into male NSG mice and 6 weeks later, three tumor compartments (pre-invasive, muscle-invasive, and super-invasive cells that had reached the superior diaphragm surface) were analyzed for differential bulk human and mouse gene expression. Whole genome transcriptomic sequencing and GO pathway analysis revealed that approximately 414 human genes were differentially expressed between the non- invasive, muscle-resident, and super-invasive tumor populations and at least 5 enriched pathways were involved. The unique expression gene patterns of muscle-resident tumor cells were reversible when compared to the pre-invasive and super-invasive expression patterns. Significant increases in g-H2AX and nuclear deformation were observed in the muscle-resident tumor clusters as compared to the non-invasive tumor mass. No differences in tumor cell proliferation, as detected by Ki67 staining, were found between the tumor clusters in the three compartments. Immunohistochemistry staining for a damage response cytokeratin (KRT6A) and integrin (CD49f) revealed heterogeneity within the muscle resident tumors as compared to the non- invasive cells. Taken together, these data suggest a hostile contractile muscle environment elicits specific responses by the invading tumor. Single cell analysis within each of the tumor compartments is currently underway to define the spatial heterogeneity of gene expression in invasive tumor cell clusters within the contractile muscle. A dynamic reciprocity of the tumor/muscle microenvironment is suggested by the analysis of the bulk transcriptomic sequencing demonstrating a significant increase in mouse muscle bio-synthetic gene transcription as a consequence of the human tumor penetrating the tissue. Taken together, these data indicate that human tumors, during the act of traversing the contractile muscle layer, respond to this unique microenvironment by transiently altering transcription, while sustaining nuclear damage which results in the reprogramming of the muscle. This new information suggests that novel tumor or muscle biomarkers might indicate early muscle invasion events and assist new high-resolution image analysis for precision diagnostic and/or therapeutic decisions. (Supported in part by P30 CA23074; F30 CA143924, UACC Team Science Award). Citation Format: Kendra D Marr, Beatrice S Knudsen, Rafael Sainz, Kelvin W Pond, Noel E Warfel, Belinda E Sun, Anne E Cress. Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A055.

Abstract Hypoxia is a physiologically relevant feature of the prostate microenvironment that promotes migration and invasion resulting in extracapsular extension, the first step toward metastatic progression. Prostate cancer invasion depends on integrins as mechanosensing membrane receptors during creation of membrane lamellipodial protrusions and focal adhesions (FAs). The objective of this study was to determine the molecular events that promote membrane protrusions under hypoxia and whether this was dependent upon kindlin-2, an essential integrin adapter that marks activated β1 integrin heterodimers. DU145 cells depleted of one copy of FERMT2+/- (50% kindlin-2 expression) by CRISPR/Cas9 or DU145 FERMT2wt (normal kindlin-2 expression) were grown under acute exposure to hypoxia (1% O2) and compared to cells grown under normal tissue culture conditions. Immunofluorescence microscopy experiments were performed to analyze the spatial temporal expression of kindlin-2 complexes. Kindlin-2 complexes were confirmed by immunoprecipitation using an anti-kindlin-2 3A3-antibody from Sigma-Aldrich. Colocalization was determined by obtaining 2D immunofluorescence microscopy images analyzed using ImageJ 2.1.0/1.53c and Nikon NIS-Elements 5.30.04. Under hypoxic conditions, analysis over four time points (4h, 8h, 12h & 16h) increased the number and area of FAs (marked by paxillin (PXN)) containing kindlin-2 in a time-dependent manner by 2-fold and 1.5-fold, respectively, but not in DU145 FERMT2+/- cells. Additionally, hypoxia increased membrane area, perimeter, and the plasma membrane intensity of kindlin-2 exclusive of FAs in DU145 FERMT2wt cells which was maximal after 8 hours of hypoxia. Interestingly, limiting the kindlin-2 expression in FERMT2+/- cells resulted in a loss of hypoxia-induced lamellipodial protrusions (marked by lamellipodin (RAPH1)) containing kindlin-2 while hypoxia-induced kindlin-2 FA changes were preserved. The current working hypothesis is that lamellipodial protrusions are dependent upon kindlin-2 expression whereas the established FAs are stable under conditions of reduced kindlin-2 expression in hypoxia. This data suggests that an early stage of migration, lamellipodial extensions are sensitive to kindlin-2 availability. Further studies are required to determine the dynamic interplay between protrusive events and focal adhesions in relation to kindlin-2 for prostate cancer cells to migrate and invade. Citation Format: Colin Nelson, Daniel Hernandez-Cortes, Kendra D. Marr, Jaime MC Gard, Allan I. Paxson, William L. Harryman, Natalya K. Seppanen, John M. Ryniawec, Anne E. Cress. Lamellipodial protrusions induced by hypoxia depend upon kindlin-2 in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5418.

Abstract Phenotype heterogeneity is a hallmark of prostate adenocarcinoma and is a major source of recurrent disease. We tested pre-clinical therapies to eradicate phenotypically distinct cell populations derived from the parental DU145WT prostate cancer cell line. The derived cell lines are called DU145AA, DU145J7, and DU145G6. The DU145AA variant is a non-aggressive cell population while the DU145J7 and DU145G6 are aggressive phenotypes that invade through muscle and metastasize to bone. Flow cytometry analysis documented the phenotypic plasticity of the DU145 cell variants since DU145 aggressive lines expressed 1.3-fold increases in matrix metalloprotease 14 surface expression and a 2-fold increase in tri-methyl Histone H3 at lysine 27 as compared to the DU145WT parent line. The DU145AA non-aggressive line expressed an epithelial cell-cell lineage phenotype with up to 2-fold increases in E-cadherin, claudin 4, claudin 7, and a unique cytokeratin 6A expression and a 2-fold loss of Zinc finger E-box-binding homeobox 1, EVL, and Kindlin 2. A collaboration with the National Center for Advancing Translational Science (NCATS) resulted in the identification of pharmacological agents active in eradicating DU145J7 and DU145G6 cells as compared to the DU145WT population. The agents were selected from a quantitative high-throughput screen of 10,677 combined investigative and approved anti-cancer agents using the Cell Titer-Glo Luminescent Cell Viability assay. We independently validated 31 compounds from the assay that have 2-fold, or greater, differences in concentration to inhibit 50% cell growth (IC50) between the wild-type and aggressive variants of DU145 cells. The top 10 hits were tested for the efficacy of different combinations to eradicate defined admixtures of aggressive and non-aggressive phenotypes, including the DU145AA cells, to model tumor heterogeneity. Tested compounds with differential sensitivity between aggressive and non-aggressive cells were those inhibiting histone deacetylase (vorinostat, fimepinostat, and pracinostat), proteasomes (bortezomib, delanzomib, ixazomib), topoisomerases (gimatecan and daunorubicin), and other compounds identified independently by us targeting nicotinamide phosphoribosyl transferase (FK866) and DNA (bleomycin). Using admixtures of tumor cell phenotypes, we determined optimal combinations of compounds that would selectively eradicate the sub-populations. The results suggest that determination of phenotypes within tumor populations may eliminate the heterogenous tumor and prevent recurrent disease. Funding was provided by the University of Arizona Cancer Center (NCI-P30 CA23074 and NCI-R01 CA159406) and by the Partnership in Native American Cancer Prevention at the University of Arizona (U54CA143924) and Northern Arizona University (U54CA143925). Collaborators at NCATS were supported by the intramural research program. Citation Format: Allan I. Paxson, Loren H. Chang, Kendra D. Marr, Jaime M. Gard, Colin S. Nelson, Abhijeet Kapoor, William L. Harryman, Juan J. Marugan, Mark J. Henderson, Tino W. Sanchez, Anne E. Cress. Increasing the therapeutic vulnerability of heterogenous cell phenotypes within prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4301.

Prostate 3D cell networks require expression of integrin α6, FAK, and uPAR

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6β1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

Specific guide RNAs and positions used for genomic editing of α6 integrin using CRISPR/Cas9 in DU145 cells injected into mice and resulting tumor growth kinetics

ECM proteolysis, α6 integrin, and E-cadherin expression in 3D Matrigel invasive networks

Pancreas invasion is independent of integrin α6 status

Invasive Network formation in DU145 α6WT cells

The advent of perpetuating living organoids derived from patient tissue is a promising avenue for cancer research but is limited by difficulties with precise characterization. In this brief communication, we demonstrate via time-lapse imaging distinct phenotypes of prostate organoids derived from patient material– without confirmation of cellular identity. We show that organoids derived from histologically normal tissue more readily spread on a physiologic extracellular matrix (ECM) than on pathologic ECM (p<0.0001), while tumor-derived organoids spread equally on either substrate (p=0.2406). This study is an important proof-of-concept to defer precise characterization of organoids and still glean information into disease pathology.

Muscle Invasive Networks in Aggressive Human Prostate Cancer

Invasive Network formation in DU145 α6AA cells

Bladder, colon, gastric, prostate, and uterine cancers originate from the organ’s epithelium, and each organ is surrounded by smooth muscle. In prostate cancer, organ-confined tumors, without extracapsular extension (ECE) through muscle, have a 5-year survival rate of 99% compared to 31% for metastatic disease. Previously, we modeled tumor cluster ECE and reported that a CRISPR-Cas9 integrin mutation (α6AA) blocked ECE by switching from a cell-ECM to a cell-cell biophysical phenotype. Since tumors are mosaics and migrate as heterogeneous drug-resistant populations, we tested the functional heterogeneity of the two biophysical cell phenotypes in ECE. Experimental procedures included the in vivo mouse xenograft model of ECE as a functional endpoint of muscle invasion, the MTT assay for cell survival, and using electric cell impedance sensing (ECIS) measurements to directly compare the biophysical properties of tumor cells expressing the invasion permissive α6WT integrin, cells expressing only the α6AA mutation, or a mixed population expressing both integrin types. The α6AA integrin mutation or α6KO knockout produced cells unable to invade into and through the smooth muscle of the mouse as compared to the α6WT integrin cells, as previously reported by us. The new unpublished results are that cell adhesion mediated drug resistance was detected in the α6AA population since their LD50 to Bortezomib, Gemcitabine, and Taxotere was shifted to 33.71nM, 64.66nM, and 5.77nM respectively from 16.29nM, 31.74nM, and 3.52nM in the α6WT cells as measured by MTT assay at 72 hours of incubation. In contrast, the α6AA cells were sensitized to the NAMPT inhibitor, FK866, with LD50 shifting from 27.46nM in α6WT to 7.354nM in the α6AA cells. A mixture of α6AA and α6WT cells allowed invasion into and through the muscle in the mouse of the mutant α6AA integrin cells that could not invade on their own. Examining the live cellular biophysical parameters revealed that the integrin mutation produced a 4-fold increase in cell-cell resistance (400Hz measurement) and a 12-fold recovery time delay in re-establishing a cell-cell resistance monolayer after wounding, with response complexity decreased two-fold. Cell-ECM resistance (40,000Hz measurement) in α6AA versus α6WT population was 1.5-fold decreased with a 6-fold recovery time delay in the cell-ECM resistance monolayer and response complexity decreased 2-fold. A heterogenous population containing up to a 4:1 proportion of α6AA to α6WT cells synergistically delayed cell-cell recovery time of the wounded monolayer up to 6-fold compared to the α6WT cells. Conversely, recovery of the wounded cell-ECM resistance monolayer with 1:1 or 4:1 mixture of α6AA cell only delayed recovery time by 1.5–1.7-fold. Taken together, these data suggest that a heterogenous tumor population provides functional advantages for drug-resistant tumor cells to invade and traverse the muscle barrier. Current work is ongoing to optimize the drug combinations to eliminate both phenotypes in the invasive tumor network and prevent ECE. Citation Format: Kendra D. Marr, Jaime M.C. Gard, William L. Harryman, Elijah J. Keeswood, Allan I. Paxson, Charles Wolgemuth, Lori A. Hazlehurst, Raymond B. Nagle, Anne E. Cress. Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B001.

In 2023, approximately 288,300 new diagnoses of prostate cancer will occur, with 34,700 disease-related deaths. Death from prostate cancer is associated with metastasis, enabled by progression of tumor phenotypes and successful extracapsular extension to reach Batson’s venous plexus, a specific route to the spine and brain. Using a mouse-human tumor xenograft model, we isolated an aggressive muscle invasive cell population of prostate cancer, called DU145J7 with a distinct biophysical phenotype, elevated histone H3K27, and increased matrix metalloproteinase 14 expression as compared to the non-aggressive parent cell population called DU145WT. Our goal was to determine the sensitivities to known chemotherapeutic agents of the aggressive cells as compared to the parent population. High-throughput screening was performed with 5,578 compounds, comprising of approved and investigational drugs for oncology. Eleven compounds were selected for additional testing, which revealed that vorinostat, 5-azacitidine, and fimepinostat (epigenetic inhibitors) showed 2.6-to-7.5-fold increases in lethality for the aggressive prostate cancer cell population as compared to the parent, as judged by the concentration of drug to inhibit 50% cell growth (IC50). On the other hand, the DU145J7 cells were 2.2-to-4.0-fold resistant to mitoxantrone, daunorubicin, and gimatecan (topoisomerase inhibitors) as compared to DU145WT. No differences in sensitivities between cell populations were found for docetaxel or pirarubicin. The increased sensitivity of DU145J7 prostate cancer cells to chromatin modifying agents suggests a therapeutic vulnerability occurs after tumor cells invade into and through muscle. Future work will determine which epigenetic modifiers and what combinations will be most effective to eradicate early aggressive tumor populations.

<p>Integrin mutant increases cell-cell biophysical features</p>

Overtreatment of prostate cancer is a significant source of patient morbidity and cost. The human prostate is bounded by a smooth muscle capsule, and aggressive tumors invade through the muscle layer, called extracapsular extension (ECE), to escape organ confinement. The presence of ECE defines pT3a pathologic stage and is associated with increased risk of biochemical recurrence, metastases, and cancer-specific mortality. Although muscle invasion is required for ECE and metastatic spread, both the invasive tumor network phenotypes and muscle responses are understudied. The goal was to probe the molecular events of ECE to understand this critical step in metastasis. Muscle invasion of prostate cancer cell lines was tested in vivo by injecting cells into the peritoneal cavity of male NSG mice. The cells colonize the inferior surface of the muscular respiratory diaphragm and invade through to the superior surface. To explore transcriptional regulators, we performed whole genome RNAseq on cells from three compartments: (1) “Inferior” non-invading cells on the underside of the diaphragm; (2) “Muscle-resident” cells that have invaded and now reside within the diaphragm muscle; and (3) “Superior” cells that have completely traversed the diaphragm. Tumors cells reaching the superior side of the diaphragm were established ex vivo as polyclonal cell lines termed the “KM” series. RNAseq reveals 1,482 differentially expressed sequences (DES) between Inferior and Muscle-resident cells, 253 DES between Muscle-resident and Superior cells, and 896 DES between Inferior and Superior cells (padj <0.05, |fold change| ≥ 1.3). Further, we identified 84 DES that overlap between the Superior vs. Muscle-resident and Inferior vs. Muscle-resident groups, highlighting candidate gene changes unique to cancer cells in a muscle microenvironment. Importantly, 4,076 differentially expressed, mouse-specific RNA sequences (padj < 0.05, |fold change| ≥ 2.0) highlight pathways within muscle that respond to the presence of invasive tumors. Strikingly, the KM series have aggressive features including newly acquired bone metastasis and resistance to a taxane. The gene expression patterns imply a dominant effect of the muscle microenvironment in evoking a new and transient tumor transcriptional response as revealed by the DESs. Independent of this response, the KM sub-populations successfully navigating the muscle were more aggressive in laboratory assays, including bone metastatic potential and chemotherapeutic resistance. These studies support the hypothesis that successful invasion into and through a contractile muscle layer results in aggressive cancer cells. Using the in vivo assay provides the unique ability to parse out unexplored patterns of changes that occur in early, muscle-invasive disease to improve prostate cancer patient selection for definitive treatment versus active surveillance. Citation Format: Kendra D. Marr, Beatrice S. Knudsen, Jaime M. Gard, Malia Bird, Raymond B. Nagle, Anne E. Cress. Muscle invasion produced drug-resistant and bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3810.

Muscle-invasive lethal carcinomas traverse into and through this specialized biophysical and growth factor enriched microenvironment. We will highlight cancers that originate in organs surrounded by smooth muscle, which presents a barrier to dissemination, including prostate, bladder, esophageal, gastric, and colorectal cancers. We propose that the heterogeneity of cell-cell and cell-ECM adhesion receptors is an important driver of aggressive tumor networks with functional consequences for progression. Phenotype heterogeneity of the tumor provides a biophysical advantage for tumor network invasion through the tensile muscle and survival of the tumor network. We hypothesize that a functional epithelial-mesenchymal cooperation (EMC)exists within the tumor invasive network to facilitate tumor escape from the primary organ, invasion and traversing of muscle, and navigation to metastatic sites. Cooperation between specific epithelial cells within the tumor and stromal (mesenchymal) cells interacting with the tumor is illustrated using the examples of laminin-binding adhesion molecules-especially integrins-and their response to growth and inflammatory factors in the tumor microenvironment. The cooperation between cell-cell (E-cadherin, CDH1) and cell-ECM (α6 integrin, CD49f) expression and growth factor receptors is highlighted within poorly differentiated human tumors associated with aggressive disease. Cancer-associated fibroblasts are examined for their role in the tumor microenvironment in generating and organizing various growth factors. Cellular structural proteins are potential utility markers for future spatial profiling studies. We also examine the special characteristics of the smooth muscle microenvironment and how invasion by a primary tumor can alter this environment and contribute to tumor escape via cooperation between epithelial and stromal cells. This cooperative state allows the heterogenous tumor clusters to be shaped by various growth factors, co-opt or evade immune system response, adapt from hypoxic to normoxic conditions, adjust to varying energy sources, and survive radiation and chemotherapeutic interventions. Understanding the epithelial-mesenchymal cooperation in early tumor invasive networks holds potential for both identifying early biomarkers of the aggressive transition and identification of novel agents to prevent the epithelial-mesenchymal cooperation phenotype. Epithelial-mesenchymal cooperation is likely to unveil new tumor subtypes to aid in selection of appropriate therapeutic strategies.