The tumor microenvironment has emerged as a novel chemotherapeutic strategy in the treatment of cancer. This is most clearly exemplified by the antiangiogenesis class of compounds. Therapeutic ...strategies that target fibroblasts within the tumor stroma offer another treatment option. However, despite promising data obtained in preclinical models, such strategies have not been widely used in the clinical setting, largely due to a lack of effective treatments that specifically target this population of cells. The identification of fibroblast activation protein α (FAP) as a target selectively expressed on fibroblasts within the tumor stroma or on carcinoma-associated fibroblasts led to intensive efforts to exploit this novel cellular target for clinical benefit. FAP is a membrane-bound serine protease of the prolyl oligopeptidase family with unique post-prolyl endopeptidase activity. Until recently, the majority of FAP-based therapeutic approaches focused on the development of small-molecule inhibitors of enzymatic activity. Evidence suggests, however, that FAP's pathophysiological role in carcinogenesis may be highly contextual, depending on both the exact nature of the tumor microenvironment present and the cancer type in question to determine its tumor-promoting or tumor-suppressing phenotype. As an alternative strategy, we are taking advantage of FAP's restricted expression and unique substrate preferences to develop a FAP-activated prodrug to target the activation of a cytotoxic compound within the tumor stroma. Of note, this strategy would be effective independently of FAP's role in tumor progression because its therapeutic benefit would rely on FAP's localization and activity within the tumor microenvironment rather than strictly on inhibition of its function.
The development of mesenchymal stem cells (MSCs) as cell‐based drug delivery vectors for numerous clinical indications, including cancer, has significant promise. However, a considerable challenge ...for effective translation of these approaches is the limited tumor tropism and broad biodistribution observed using conventional MSCs, which raises concerns for toxicity to nontarget peripheral tissues (i.e., the bad). Consequently, there are a variety of synthetic engineering platforms in active development to improve tumor‐selective targeting via increased homing efficiency and/or specificity of drug activation, some of which are already being evaluated clinically (i.e., the good). Unfortunately, the lack of robust quantification and widespread adoption of standardized methodologies with high sensitivity and resolution has made accurate comparisons across studies difficult, which has significantly impeded progress (i.e., the ugly). Herein, we provide a concise review of active and passive MSC homing mechanisms and biodistribution postinfusion; in addition to in vivo cell tracking methodologies and strategies to enhance tumor targeting with a focus on MSC‐based drug delivery strategies for cancer therapy. Stem Cells Translational Medicine 2018;1–13
Mechanical barriers resulting from small vessels in the vascular network are a significant impediment to MSC trafficking via systemic circulation, which severely limits access of exogenously‐introduced MSCs to many target tissues, including tumors, and has implications for MSC‐based drug delivery strategies.
Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, ...secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4-induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8
T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. SIGNIFICANCE: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression.
Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a ...post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG.
The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided.
FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5 nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206 mm(3), 95% CI = 0.068 to 0.344 mm(3); control: mean = 0.580 mm(3), 95% CI = 0.267 to 0.893 mm(3)) and 0.24 (treated: mean = 0.131 mm(3), 95% CI = 0.09 to 0.180 mm(3); control: mean = 0.543 mm(3), 95% CI = 0.173 to 0.913 mm(3)), respectively, on day 21 after therapy.
This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.
The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd-generation androgen receptor ...signaling inhibitors (ARSi) like abiraterone acetate (Abi) and enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi resistance. Elevated expression of AR-FL alone is sufficient for Abi but not Enza resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL- and AR-V7-dependent transcriptional complementation are needed for Abi/Enza resistance.
Background
Prostate cancer is characterized by T‐cell exclusion, which is consistent with their poor responses to immunotherapy. In addition, T‐cells restricted to the adjacent stroma and benign ...areas are characterized by anergic and immunosuppressive phenotypes. In order for immunotherapies to produce robust anti‐tumor responses in prostate cancer, this exclusion barrier and immunosuppressive microenvironment must first be overcome. We have previously identified mesenchymal stem cells (MSCs) in primary and metastatic human prostate cancer tissue.
Methods
An Opal Multiplex immunofluorescence assay based on CD73, CD90, and CD105 staining was used to identify triple‐labeled MSCs in human prostate cancer tissue. T‐cell suppression assays and flow cytometry were used to demonstrate the immunosuppressive potential of primary MSCs expanded from human bone marrow and prostate cancer tissue from independent donors.
Results
Endogenous MSCs were confirmed to be present at sites of human prostate cancer. These prostate cancer‐infiltrating MSCs suppress T‐cell proliferation in a dose‐dependent manner similar to their bone marrow‐derived counterparts. Also similar to bone marrow‐derived MSCs, prostate cancer‐infiltrating MSCs upregulate expression of PD‐L1 and PD‐L2 on their cell surface in the presence of IFNγ and TNFα.
Conclusion
Prostate cancer‐infiltrating MSCs suppress T‐cell proliferation similar to canonical bone marrow‐derived MSCs, which have well‐documented immunosuppressive properties with numerous effects on both innate and adaptive immune system function. Thus, we hypothesize that selective depletion of MSCs infiltrating sites of prostate cancer should restore immunologic recognition and elimination of malignant cells via broad re‐activation of cytotoxic pro‐inflammatory pathways.
Tumor-associated macrophages (TAMs) are an abundant tumor-promoting cell type in the tumor microenvironment (TME). Most TAMs exhibit a pro-tumor M2-like phenotype supportive of tumor growth, immune ...evasion, and metastasis. IL-4 and IL-13 are major cytokines that polarize macrophages to an M2 subset and share a common receptor, IL-4 receptor alpha (IL-4R alpha). Treatment of human ex vivo polarized M2 macrophages and M2 macrophage precursors with IL-4R alpha antagonist antibody Dupilumab (DupixentⓇ) reduces M2 macrophage features, including a shift in cell surface marker protein expression and gene expression. In animal models of prostate cancer, both pharmacologic inhibition of IL-4R alpha and genetic deletion of IL-4R alpha utilizing an Il4ra -/- mouse model result in decreased CD206 on TAMs. These data support IL-4R alpha as a target to reduce the pro-tumor, M2-like macrophage phenotype as a novel adjunct cancer therapy.
Background
Presently, ∼85 serially transplantable human prostate cancer xenografts spanning the phenotypic, epigenetic, and genetic heterogeneity seen clinically are available in a variety of ...laboratories throughout the world. If distributed to the prostate cancer research community, these can provide an experimental platform for resolving the specificity versus generalizability of basic cancer biology principles (eg, credentialing of therapeutic molecular targets) and for validating translational approaches for prevention, diagnosis, and therapy. Thus, there is an urgent need to distribute the already established serially transplantable human prostate cancer xenografts and to develop robust methods for establishing new ones.
Methods
To accelerate the development of such additional xenografts, particularly from patients treated with the newer standard of care agents (ie, abiraterone, enzalutamide, cabazitaxel, alpharadin, etc), a historic review of the field will be presented.
Results
Over the last 50 years, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer xenografts using a variety of immune deficient mice. These are summarized chronologically.
Conclusions and Future
With the ever growing appreciation of the value of personalized medicine (aka precision medicine), methods need to be developed that allow efficient and timely growth of primary patient derived prostate cancer xenografts (PDXs), which can be used as “avatars” for defining optimal therapy for that specific patient. Such development should be based upon the leads obtained from the successful establishment of serially transplantable prostate cancer xenografts described in this review.
Mesenchymal stem cells (MSCs) have an inherent tropism for sites of inflammation, which are frequently present in sites of cancer, including prostatic lesions. MSCs have been defined as ...CD73/CD90/CD105 triple-positive cells in the absence of hematopoietic lineage markers with the ability to differentiate into multiple mesodermal lineages, including osteoblasts, adipocytes, and chondrocytes. Our group has previously demonstrated that MSCs represent between 0.01 and 1.1% of the total cells present in human prostatectomy tissue. In addition to their multi-lineage differentiation potential, MSCs are immunoprivileged in nature and have a range of immunomodulatory effects on both the innate and adaptive arms of the immune system. MSCs have been detected in an increasing array of tissues, and evidence suggests that they are likely present in perivascular niches throughout the body. These observations suggest that MSCs represent critical mediators of the overall immune response during physiological homeostasis and likely contribute to pathophysiological conditions as well. Chronic inflammation has been suggested as an initiating event and progression factor in prostate carcinogenesis, a process in which the immunosuppressive properties of MSCs may play a role. MSCs have also been shown to influence malignant progression through a variety of other mechanisms, including effects on tumor proliferation, angiogenesis, survival, and metastasis. Additionally, human bone marrow-derived MSCs have been shown to traffic to human prostate cancer xenografts in immunocompromised murine hosts. The trafficking properties and immunoprivileged status of MSCs suggest that they can be exploited as an allogeneic cell-based vector to deliver cytotoxic or diagnostic agents for therapy.
Imaging Mass Cytometry (IMC) is an emerging multiplexed imaging technology for analyzing complex microenvironments using more than 40 molecularly-specific channels. However, this modality has unique ...data processing requirements, particularly for patient tissue specimens where signal-to-noise ratios for markers can be low, despite optimization, and pixel intensity artifacts can deteriorate image quality and downstream analysis. Here we demonstrate an automated content-aware pipeline, IMC-Denoise, to restore IMC images deploying a differential intensity map-based restoration (DIMR) algorithm for removing hot pixels and a self-supervised deep learning algorithm for shot noise image filtering (DeepSNiF). IMC-Denoise outperforms existing methods for adaptive hot pixel and background noise removal, with significant image quality improvement in modeled data and datasets from multiple pathologies. This includes in technically challenging human bone marrow; we achieve noise level reduction of 87% for a 5.6-fold higher contrast-to-noise ratio, and more accurate background noise removal with approximately 2 × improved F1 score. Our approach enhances manual gating and automated phenotyping with cell-scale downstream analyses. Verified by manual annotations, spatial and density analysis for targeted cell groups reveal subtle but significant differences of cell populations in diseased bone marrow. We anticipate that IMC-Denoise will provide similar benefits across mass cytometric applications to more deeply characterize complex tissue microenvironments.
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