Penile cancer (PeCa) represents 0.2% of all cancers diagnosed worldwide (1). However, poor and developing countries have a high incidence of the disease (2–4). Partial penectomy is frequently used for localized carcinomas (5). The disease could be aggressive, metastatic, and mutilating, mainly due to the delay in seeking treatment (6). Despite all efforts to improve the therapeutic strategies, the survival rates of PeCa patients remained almost unchanged over the past years (7).

Molecular and functional studies have revealed an important role of cells composing the tumor microenvironment (TME) in PeCa. The presence and distribution of immune checkpoint molecules or immune cell components were shown to be a potential predictor of clinical outcomes [reviewed in Aydin et al. (8)]. However, the immune fraction of TME alone is insufficient to predict treatment response and survival (8). Cancer-associated fibroblasts (CAFs) are a key component of the TME, playing a critical role in the extracellular matrix (ECM) deposition and remodeling. Moreover, CAFs have been implicated in the modulation of the immune system by establishing an immunosuppressive stroma, which can promote resistance to immune-based therapies (9). Although a limited number of transcriptome analyses in PeCa has been reported (7), the enrichment of pathways associated with ECM organization was described in patients with lymph node (LN) metastasis (10).

Immunotherapeutic drugs inhibit the immune checkpoint genes such as programmed cell death 1 (PD-1) and its ligand (PD-L1) (11). Cocks et al. (2016) identified PD-L1 expression in approximately 40% of PeCa patients, who may benefit from immunotherapies (12). Immunotherapy was further supported by studies that found that most patients presented advanced cancer (12, 13). The remaining 60% of PeCa patients have limited therapeutic options, including organ amputation and standard-of-care chemotherapies. In these cases, the immunotherapy response could be enhanced using a combinatorial treatment with TME-modulating drugs (14). Targetable molecular mechanisms that modulate CAFs are suggested to increase the cytotoxic T-cell level in the tumor, contributing to an increased immunotherapy response (15). Despite efforts to characterize the immune environment in PeCa (8, 13, 16, 17), there is a lack of in-depth knowledge on how the immune cells and CAFs simultaneously affect tumor progression.

The transcriptome analysis of bulk tumor samples allows in silico deconvolution using computational tools to infer cell type proportions (18). Moreover, the tumor transcriptome profile of the secretome (genes encoding secreted proteins) indicates which cell is activated in the tumor and releases factors that allow communication with other cells (19–21). These strategies are valuable tools to identify enriched cell types within the TME and their contribution to the tumor progression and response to therapy.

Here, we explored the transcriptome from two perspectives: 1) identify enriched immune and stromal cells using an in silico deconvolution method and 2) investigate targetable secretome components for TME modulation in PeCa. These strategies allowed us to characterize the TME composition of PeCa, in which we verified an enrichment of CAFs inversely correlated with immune cell proportion and an association with poor survival. Once the TME was characterized, the next step was to evaluate genes associated with ECM remodeling to identify potential drug targets able to modulate CAFs. Among these genes, we confirmed high expression levels of matrix metalloproteinase (MMP) genes in PeCa samples. Using PeCa–derived CAFs, we inhibited MMP expression and demonstrated a low viability of the cells.

The patients included in this study showed similar clinical and histopathological characteristics, such as mean age, alcohol consumption, tobacco usage, HPV status, TNM stage, and perineural and angiolymphatic invasion ( Table S1 ).

We identified 2,199 and 1,050 upregulated genes in PeCa compared with normal tissues in our internal and validation datasets, respectively, of which 161 and 189, respectively, were predicted to encode secreted proteins. The PPI analysis of the secretome genes revealed functions associated with ECM and inflammation ( Figures 1A, B ). Seventeen terms with the highest combined score were mainly associated with inflammatory response and ECM regulation in both PeCa datasets ( Table S3 ; Figure 1C ). Despite enriching similar pathways and ontologies, only 23 secretory genes (encoding inflammatory cytokines/chemokines and ECM molecules) were upregulated in internal and validation datasets ( Table 1 ). These findings suggested that PeCa cells directly interact with the immune system and the stroma.

In this study, we explored the transcriptome data of PeCa samples to evaluate the interplay between cells within the TME and its relevance to disease outcomes. We identified an enrichment of immune and stromal cells and an association with survival. A second and complementary approach was based on investigating targetable MMPs for TME modulation in PeCa.

We found that immune cells and CAFs play a critical role in the TME by expressing and potentially secreting inflammatory factors and ECM remodeling proteinases. We also verified that immune cell proportions were negatively correlated with CAFs in PeCa samples. Interestingly, patients with high CAF scores presented lower survival rates and an increased expression of MMPs and collagens. These results demonstrate that our strategy to profile and deconvolute bulk tumors brings new perspectives to understand the TME of PeCa better. These findings also provided the rationale to test, in vitro, the MMP inhibitor GM6001 on PeCa-derived CAFs. We observed a higher effect of this inhibitor in penile CAFs than in normal fibroblasts.

Extracellular components and inflammatory factors were the main class of upregulated secreted proteins found in our internal and validation PeCa datasets. We found 23 secretome genes shared in these two datasets. This small overlap could be explained by the different microarray platforms used or simply by the intrinsic heterogeneity found in cancer samples. However, enriched pathways and gene ontologies were mainly associated with extracellular matrix and immune response in both datasets, reinforcing their relevance to the disease despite the differences in the overlapping secretome. The immune-inflammatory system and matrix metalloproteases were previously demonstrated to be overrepresented in PeCa compared to normal penile tissues (10, 43). In oral carcinomas, high levels of pro-inflammatory cytokines affect the TME by increasing ECM degradation via MMPs during disease progression (44). Our findings suggest that interactions between inflammation and matrix remodeling have a crucial role in penile carcinogenesis and progression.

We found increased scores of B cells, macrophages, and DCs in PeCa compared to normal tissues. Moreover, we described that a subset of PeCa patients presented an immune hot phenotype (higher scores of CD8 in T cells, DC, and mast cells). These features are potentially associated with a better prognosis. Although the low number of our cases precluded statistical significance, B cells and DCs improve prognosis in cancer patients due to the antitumor activity and the potential to increase immunotherapy response (45–48). The immune hot score classification predicts a better prognosis in cancer patients (29). Altogether, the immune classification of PeCa could be used as a tool to predict the outcome and immunotherapy response, mainly because we also found a negative correlation between immune and CAF scores in PeCa. These results open new scenarios to test whether immunotherapy response could be enhanced using a combinatorial treatment with TME-modulating drugs.

The CAF score is increased in PeCa compared to normal samples, but we also found a subset of PeCa highly expressing CAF markers with lower overall survival (internal and validation sets). It has been established that CAF gene signatures can distinguish between low and high CAF tumors and predict patient survival (49, 50). The impact of CAFs on patients’ survival has been reported, and their inhibition has emerged as a promising anti-cancer therapy (51). However, the pharmaceutical inhibition of CAFs expressing the canonical marker FAP (fibroblast activation protein) has not been proven to be successful yet (51). CAFs contribute to an immunosuppressive TME and targeting CAFs, or their products have the potential to improve current immunotherapy approaches for cancer patients (42). Therefore, a comprehensive understanding of CAF markers is needed, aiming to design effective therapeutic strategies for PeCa.

We showed that PeCa presented a global alteration of MMPs and collagens, in which tumors with high CAF scores have an increased expression of collagens. COL11A1 was associated with LN metastasis, corroborating with previous findings (52, 53), and a novel prognostic biomarker of PeCa. Collagens are the most abundant ECM component, increasing tumor tissue stiffness, among other features (54). MMPs are essential to degrade collagen during ECM remodeling (41). A previous study demonstrated that MMP1 and MMP12 presented increased expression in usual and mixed PeCa subtypes (10). Herein, we confirmed these alterations and found an increased expression of MMP1 in tumors with high CAF scores. Epithelial cells express MMPs (55); thus, the inhibition of MMP must modulate the microenvironment and malignant epithelial cells. We showed that MMPs are highly expressed in PeCa cells and PeCa-derived CAFs. The implication of MMPs in tumor invasion and metastasis has prompted the development of strategies that promote MMP inhibition (56). A high expression of MMP1 has been related to poor outcomes and shorter overall survival in PeCa (10). CAFs express MMPs that assist the immunosuppression of TME, counteracting CAFs that secrete MMPs, which have the potential to enhance the efficacy of immunotherapies (42). Therefore, MMP inhibition is a potential therapeutic strategy for PeCa, especially in combination with standard-of-care therapies.

PeCa–derived cell lines were previously described as reliable models to investigate the molecular mechanisms associated with carcinogenesis and treatment resistance and to develop effective treatment strategies (57). Targeting therapies enabled personalized approaches to improve the outcome of PeCa patients (7, 58). The genomic profiling of PeCas revealed the potential of Epidermal Growth Factor Receptor (EGFR) target therapy, in which tumors with EGFR amplification could be more sensitive (59). However, the number of preclinical studies in PeCa is still limited. Genetically engineered mouse models of PeCa were elegantly evaluated, showing that a combined target therapy and immunotherapy could be used in the treatment of PeCa patients (17).

In the present study, to better investigate the behavior of CAFs and the therapy response, we showed that the inhibition of MMPs using a broad-spectrum MMP inhibitor presented a modest effect in PeCa-derived CAFs (2 out of 3 cells) and no effect in normal fibroblasts. The slight alteration on cell viability was not surprising; the TME remodeling does not necessarily require CAFs to die but is often associated with the modulation of CAF functions (60). Cell4 had higher MMP inhibitor sensitivity than the other cells, which could be explained by the high expression of MMP7 and MMP9 (targets of GM6001). High levels of MMP7 are associated with shorter survival in cancer patients, while the prognostic role of MMP9 is controversial (61). Cell6 presented the higher expression levels of MMP1 and MMP12 (direct targets of GM6001) and showed a better response to MMP inhibition. Thus, the MMP modulation in the TMEs needs to be individually evaluated in different tumor types to design suitable MMP targeting therapies (61). Several clinical trials have tested MMP inhibitors during the last decades, and most of these studies failed due to the lack of efficacy and severe side effects (56). As more selective inhibitors of MMPs are now available, MMP targeting could be reconsidered for cancer therapy (56). Clinical trials with new MMP inhibitors and combined therapies should be undertaken to improve therapy efficacy for PeCa patients. Considering that extensive degradation of ECM proteins via MMPs promotes tumor invasion and metastasis (62), it is crucial to remodel the ECM for the most effective treatment. Therapeutic strategies targeting aberrant ECM components for cancer treatment can act as an adjuvant for conventional chemotherapy and immunotherapy (63).

Herein, we highlighted for the first time the role of CAFs and the interplay of cells within the TME in PeCa; however, this retrospective study also has limitations, including the small sample size. We overcome this limitation by validating the gene expression findings in an independent dataset. Additional studies are necessary to validate the computational prediction of cell proportions in the TME and its prognostic impact, such as single-cell RNA sequencing studies. Translational research for PeCa is still a challenge, but recent advances in PeCa patient–derived tumor xenografts demonstrate the potential of this model to design a personalized treatment considering the genomic and TME profiling (64).

Our data highlight the interplay between cell types in the TME of penile carcinomas. We demonstrated the complexity of the TME and the association between immune cells and CAFs as a prognostic factor for PeCa patients. We found a global deregulation of collagens and MMPs and tested CAF cell lines using an MMP inhibitor, which proved the ability to modulate these cells. These findings pave the way for future studies to understand the impact of TME-modulating therapies in PeCa patients.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The studies involving human participants were reviewed and approved by the Human Research Ethics Committee from A.C.Camargo Cancer Center and Barretos Cancer Hospital, São Paulo, Brazil (Protocols 1884/14 and 1030/2015, respectively). The patients/participants provided their written informed consent to participate in this study.

SR: Concept, design, and supervision. HK, JJM, and JPS: Conduction of the experiments. SC, JSS, and FM: Bioinformatic analysis. SC, HK, and SR: Writing and editing the manuscript. AL, CS-N, and EF: Assistance with sample collection and clinical data. J-MD: Design and supervision of the drug assays. All authors: Data analysis and interpretation, reading, and approval of the final manuscript.

This work was supported by the National Institute of Science and Technology in Oncogenomics (São Paulo Research Foundation – FAPESP: #2008/57887-9 and the National Council for Scientific and Technological Development – CNPq: #573589/08-9), and the Research Council of Lillebaelt Hospital, Denmark.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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