Uterine endometrial adenocarcinoma (uADC) is the fourth most common cancer among women in the Western world, with an estimated incidence of 10–20 per 100,000 women. Although prognosis is favorable for patients identified with low-grade tumors and early-stage disease, outcomes for patients with high-grade and metastatic/recurrent tumors remain poor (Ferlay et al., 2010) and represent a therapeutic challenge. The World Health Organization (WHO) tumor classification distinguishes several histopathological types of uADC, particularly based on microscopic appearance (Cree et al., 2020): (1) endometrioid carcinoma, low grade (grades I–II) or high grade (grade III), (2) serous carcinoma, (3) clear cell carcinoma, (4) mixed carcinoma, (5) undifferentiated carcinoma, (6) carcinosarcoma, (7) neuroendocrine carcinomas, and (8) other unusual types. These types have different histological and molecular features, precursor lesions, and natural history (Matias-guiu et al., 2001; Yeramian et al., 2013). They are also stratified by Tumor Cancer Genome Atlas (TCGA)-based molecular classification into four risk groups combining gene encoding DNA polymerase ε (POLE) mutational analysis with IHC analysis of p53 and mismatch repair (MMR) proteins (PMS-2 and MSH-6) (Getz et al., 2013). This provides additional prognostic information to complement the microscopic features.

Uterine leiomyosarcoma (uLMS) accounts for 1% of all uterine malignancies, with an annual incidence rate of 0.4–0.9 per 100,000 women. Like other forms of sarcoma, uLMS spreads to other parts of the body via the bloodstream rather than the lymphatic system. Although rare, this cancer can be extremely aggressive and is known to be generally unresponsive to radiation or chemotherapy. Patient survival is highly dependent on speed of diagnosis and treatment, falling to 14% at 5 years post-diagnosis for metastatic uLMS. From a molecular viewpoint, uLMS contains complex karyotypes, with numerous chromosomal aberrations and frequent deletions affecting chromosomal arms 2p, 2q, 10q, and 13q, as well as amplifications on 1p, 5q, and 8q, and precise alterations in TP53, RB1, PTEN, MED12, YWHAE, and VIPR2 (Cuppens et al., 2018).

There is growing interest in understanding the molecular features involved in myometrial invasion, a highly valuable parameter due to its direct association with poor prognosis and limited therapeutic response in uADC and uLMS. From a histopathological perspective, a variety of invasion patterns frequently coexist in different areas of the same tumor (Euscher et al., 2013). Moreover, several tumor types show specific invasion patterns not seen in other types of cancer. These differences can be explained by the fact that cancer cell invasion is currently viewed as an adaptive and heterogeneous process (Friedl and Alexander, 2011) involving key processes such as cytoskeleton dynamics, cell adhesion plasticity, and mechanotransduction of external stimuli.

Type-specific new stroma generated at the site of active tumor invasion, the invasive tumor front (ITF), is crucial in tumor growth and invasion processes (Provenzano et al., 2006; Giatromanolaki et al., 2007). In fact, high proliferation rates and cell cycle derailment have been shown at the ITF in uADC (Horrée et al., 2007). Structures surrounding tumors such as mature collagen and smooth or striated muscle can act as a barrier to tumor invasion, as shown by collagen organization at the ITF of oral squamous cell carcinomas (Devendra et al., 2018). However, tumor cells can disrupt the continuity of such structures by remodeling the immediate stroma of the tumor to carve out paths for invasion. Indeed, invasive tumor growth in pancreatic ductal adenocarcinoma is mediated via matrix remodeling metalloproteinases (Croft et al., 2004; Rath et al., 2017). Changes in the fibrillar pattern of adjacent stroma of tumor tissues have been also observed in skin tumor (Stenbäck et al., 1999), oral squamous cell carcinoma, and lymph nodes with metastasis, among others (Yinti et al., 2015; Kardam et al., 2016). In fact, the tumor-derived extracellular matrix (ECM) is biochemically distinct in its composition and is stiffer than normal ECM. This new associated ECM compartment, rich in cross-linked collagen III (reticular fibers), has been proposed as a prospective marker of early stromal invasion in incipient tumors such as breast cancer (Sivridis et al., 2004; Acerbi et al., 2015). In addition, not only the ECM collagen composition but also the orientation of the fibers have been proposed as a prognostic signature for survival in breast cancer (Conklin et al., 2011; Xi et al., 2021).

The complex interaction between heterogenic immune cell subpopulations and tumor cells may drive tumor progression, metastasis, and resistance to therapy (Galli et al., 2020). The intensifying development of immunotherapeutic strategies calls for a better understanding of tumor-immune subpopulation interactions and spatial distribution at the ITF (Blomberg et al., 2018; König et al., 2019). In this context, the use of a multiplex immunolabeling panel is essential as it enables different cell subpopulations to be identified in one tissue section (Gorris et al., 2018). Multiplexed analysis can also simultaneously measure the expression of distinct markers in a single cell as well as spatial associations between immune cell subpopulations. We applied this technology to evaluate the complex immune environment of the ITF of uADC and uLMS. A previously developed and validated multiplex immunolabeling panel was used to simultaneously assess the phagocytic cell marker CD68 of macrophages, CD3 + and CD8 + T cells, and CD20 + B lymphocytes in a single FFPE tissue (Abengozar-Muela et al., 2020; Salas-Benito et al., 2021).

Changes in activated-leukocyte cell adhesion molecule (ALCAM) expression at the endometrial tumoral cell surface (Devis et al., 2018) and increased expression of cytoplasmic Cyclin D1 (Fusté et al., 2016) have been reported to allow dissemination and invasion of endometrial neoplastic cells. Epigenetic mechanisms play an important role in regulating gene expression during many biological processes (Sharma et al., 2009). DNA methylation is the most widely studied epigenetic modification, produced by adding a methyl group (CH3) to the 5’ carbon of cytosines in cytosine–phosphate–guanine (CpG) dinucleotides to generate 5-methylcytosine (5mC) (Bao-Caamano et al., 2020). Deregulation of this epigenetic mechanism has major implications for cancer development and progression (Diaz-Lagares et al., 2016). In this context, recent genome-wide analyses have revealed striking alterations in the methylation profile of uterine uADC and uLMS (Zhang et al., 2014; Kommoss et al., 2020; Vargas et al., 2021).

Cancers show a clear coevolution between tumor cells and the tumor microenvironment. Specific differences in the tumor microenvironment at different locations may play a role in tumor growth, metastatic progression, and therapy responses (Oliver et al., 2018; Zhang and Yu, 2019). In fact, tumor invasion is a dynamic process facilitated by bidirectional interactions between tumor cells and the microenvironment, being particularly intense at the ITF. Unlike tumor cells, tumor microenvironment elements are genetically stable and thus represent an attractive therapeutic target. Uterine uLMS and uADC are different tumor types occurring in the same organ, both of which infiltrate the myometrium during local progression. The main objective of the present study is to analyze several aspects of microenvironment response at the ITF in these two tumor types, looking for similarities and differences that could provide insight into potential new common therapeutic approaches, an aim that to our best knowledge has not previously been addressed.

In the uterus, uADC and uLMS account for a significant proportion of malignant tumors associated with distant metastasis. These tumors possess distinct morphological and molecular features, and also a differing cell of origin. However, both occur in the uterus, and each of them invades the myometrium as the initial step for tumor progression. Although dissimilar in many aspects, it is feasible that they share some adaptive responses while invading the myometrium, or alternatively, they could produce a similar response to tumor invasion in the myometrium.

The process of stromatogenesis refers to the formation of new stroma in the active sites of tumor invasion and metastasis. It is proposed that the formation of this new stroma is generated and governed by the interplay between tumor cells and the complicity of adjacent activated fibroblasts. This new stroma disrupts the normal structures of the ECM and its continuity and favors easy penetration by tumor cells, thus helping their migration and immune cell infiltration among other cells (Giatromanolaki et al., 2007; Horrée et al., 2007; Bremnes et al., 2011). The ECM composition and spatial organization of collagen fibers in the newly formed stroma is decisive not only for development of several neoplasia but for tumor prognosis (Kondo, 2011; Acerbi et al., 2015; Devendra et al., 2018; Xu et al., 2019). In fact, like type I collagen, reticulin distribution is increased in many cancers such as head and neck squamous cell cancer, breast, pancreas, and colorectal cancer (Nissen et al., 2019) and increases proliferation, migration, and metastasis in pancreatic and glioblastoma cancer cells as well as in invasive prostate cancer (Chintala et al., 1996; Menke et al., 2001; Kanematsu et al., 2004). Thus, in-depth characterization of morphometric features of reticular fibers (collagen type III) in invasive tumors could provide advanced insight into mechanisms underlying the invasion and galvanize targeted strategies for precision medicine (Kanner et al., 2013; Walke and Bhagat, 2017).

Although several aspects of the ITF have been characterized in other types of tumors, comparison between them is very complicated due to differing quantification methods. Furthermore, the exact size of the true invasion front is also under debate (Horrée et al., 2007) and little is known about the histomorphometry of the fibers at the ITF of uADC and uLMS. When analyzing the 5 × 4-mm ITF areas of uADC and uLMS, we found very few significant differences between morphometric parameters, indicating a striking similarity between the ITF of the two tumor types. In a restrictive analysis, choosing a uADC sample group with a more homogeneous architecture (based on subjective criteria), all the significant differences disappeared, suggesting that an inclusive analysis reflects the architectural heterogeneity of the uADC tumors. We also circumvented the issue of variable tissue amounts when analyzing 5 × 4-mm ITF ROIs by increasing the magnification of the ROIs to 1 mm² within the previously studied 5 × 4-mm ROIs in which we ensured 50% each of the tumor and myometrium. After the necessary controls, we found that the area of the fibers in the 1-mm² ITF was no longer significantly different between uADC and uLMS, but the differences in deformity remained, indicating the potential value of this parameter as a differential characteristic between the two types of tumors. We hypothesized that the area of the fibers in the 5 × 4-mm areas could vary due to an uneven contribution of the tumor to the analysis, given that the area was no longer different in the 1-mm² ITF ROIs. The algorithm showed notably high sensitivity when measuring morphometric parameters, since in the 1-mm² areas of myometrium we did not observe any significant differences between the two tumors, as expected. However, the large number of differences observed between 1-mm² areas of pure tumor tissue reflects the ability to detect differences between negligible reticular fiber features of the tumors under subjective criteria.

At a microscopic level, ROIs of 1 mm² were sufficient for comparison of reticular fiber morphometric parameters between the tumor, myometrium, and ITF of uADC and uLMS. Furthermore, 1-mm² ROIs allowed us to characterize the microarchitecture of the reticular fibers within the same type of tumor. Interestingly, comparisons between tumor, myometrium, and ITF in uLMS revealed a high degree of similarity and little disruption of tissue architecture. Conversely, in uADC we observed a very high number of significantly different morphometric features comparing the abovementioned ROIs (Table 1), suggesting that continuity of ECM architecture is much more disrupted in uADC than uLMS tumors. Regardless of statistical difference, observing the mean of morphometric parameters at the ITF of uADC, we found that almost all values fell within the range of myometrium and tumor means, except for density (along with increased%SA) and aspect (linearization) of fibers (Table 1). Since the areas were carefully chosen to represent 50% each of myometrium and tumor components, we hypothesized that most changes observed at the uADC ITF could be attributable to the contribution of adjacent tissue. However, we cannot exclude the possibility of orchestrated ECM remodeling by tumor and non-tumoral cells to favor tumor cell migration (Giatromanolaki et al., 2007; Horrée et al., 2007; Bremnes et al., 2011). In fact, during tumor progression, cancer-associated fibroblasts are key players in dysregulated collagen turnover, leading to tumor fibrosis as evidenced by increased collagen depositions in the immediate periphery of the tumor (Pankova et al., 2016). Consistent with our results, we observed an increase of fiber density at the uADC ITF compared to tumor. In addition, the collagens are often cross-linked and linearized leading to stiffening of the tissue in several tumors (Nissen et al., 2019), which we also observed in uADC ITF as displayed by a significantly longer appearance (linearization) of the reticular fibers at the uADC ITF. ECM stiffening could in turn elicit behavioral effects on surrounding tumor cells regarding cell proliferation, differentiation, gene expression, migration, invasion, metastasis, and survival, all hallmarks of cancer (Pickup et al., 2014), and which correlate with worse patient outcomes patient (Leeming et al., 2011; Kehlet et al., 2016). Nevertheless, there are still several features of the fiber topology to take into account for future studies that would require smaller regions, such as analysis of fiber orientation with respect to the tumor–myometrium boundary, which would provide additional value for prognosis (Acerbi et al., 2015; Conklin et al., 2011; Xu et al., 2019).

Through multiplex cellular analyses, we could demonstrate that the uADC ITF contains a heterogeneous immune environment compared to the one in LSM. We found variance between different CD8 + T cell and macrophage subpopulations of different ITF in uADC and LSM. This suggests diverse myeloid and lymphocyte compositions of ITF immune environments, with CD8 + T cell and macrophage density possibly influenced by the presence of certain ECM components, such as collagens fibers, which could prevent interactions with other immune cells.

It has been shown that the reciprocal interaction between the ECM and tumor and non-tumor cells such as myofibroblasts determines recruitment, activation, and reprogramming of stromal, inflammatory, and immune cells (Sanegre et al., 2020). Despite the great similarity of reticulin scaffolding at the ITF of uADC and uLMS, we observed a differential immune infiltrate pattern, as informed by CD20, CD3, and CD8 markers. We thus cannot attribute a direct effect of reticulin architecture on the immune infiltrate composition right at the ITF. However, there could be a relationship between the morphometry of reticular fibers and the total amount of infiltrate, since the immune population is the same in both tumor types. A dense architectural barrier can be imposed not only by reticular fibers but also by ECM elements such as other collagens, hyaluronic acid, and laminins. Whereas high molecular weight hyaluronic acid provides structural integrity and suppresses the immune response by increasing regulatory T cell activity, laminins prevent transmigration and polarize leukocytes (Bollyky et al., 2009; Chanmee et al., 2016; Simon and Bromberg, 2017). ECM remodeling enzymes such as metalloproteinases, matrikines, and versicans act as cytokines and chemokines promoting IL expression and T cell chemoattractants, polarizing and activating the immune cells (Thomas et al., 2007; Hope et al., 2016). In fact, although an upregulation of collagen genes in uLMS tumors can be observed, it is still not clear whether these genes are responsible for immune density distribution. Another plausible explanation for leukocyte accumulation at the ITF of uADC could be that reticulin morphometric description suggests that uADC tumor areas seem stiffer than uLMS tumors, as indicated by the larger area and width and greater branching and fractal dimension of the fibers (although fewer fibers can be counted). Stiffer tumors could lead to a physical barrier, thus increasing the leukocyte concentration at the ITF. In addition, ECM stiffness can induce chromosomal rearrangements and transcriptional changes in several diseases (Simi et al., 2018; López-Carrasco et al., 2020) and many stiffness-sensitive genes may respond to stiffness in non-linear ways (Darnell et al., 2018).

Transcriptomic profiling of the ITF of uADC and uLMS suggests that activated neutrophils secreting antimicrobial peptides (AMP) may play a role in the immune response to oncogenesis in the context of endometrial tumors. Indeed, antimicrobial humoral response seems to be inactivated in uLMS, in stark contrast to uADC, in which there is an upregulation of this signature. AMPs have been described as important actors in angiogenesis and modulation of the immune response, via stimulation of chemokines and chemotaxis of leukocytes, and may also exert cytotoxic activity against tumor cells (Al-Rayahi and Sanyi, 2015; Jin and Weinberg, 2019). In fact, LCN2, one of the top upregulated genes in the ITF of uADCs (Supplementary Table 2), codes for neutrophil gelatinase-associated lipocalin, which has been reported to be associated with aggressive features of endometrial uADC (Mannelqvist et al., 2012; Cymbaluk-PA Oska et al., 2019) but is also described as an invasiveness and metastasis suppressor in other contexts (Lee et al., 2006; Lim et al., 2007; Tong et al., 2008; Santiago-Sánchez et al., 2020). Another highly upregulated antimicrobial peptide, DEFB1 (Supplementary Table 2), belongs to a family of cytotoxic peptides made by neutrophils. This gene has recently been identified as a chromosome 8p tumor-suppressor gene, downregulated in several malignancies such as salivary gland tumors, oral squamous cell carcinoma, and colon and liver cancer (Álvarez et al., 2018; Sun et al., 2019). Loss of orthologous murine defense-1 in mice enhances nickel sulfate-induced uLMS and causes mouse kidney cells to exhibit increased susceptibility to HPV-16 E6/7-induced neoplastic transformation (Sun et al., 2019). DEFB1 also induces formation of neutrophil extracellular traps (NETs) (Álvarez et al., 2018), mesh-like structures composed of cytosolic and granule proteins which are assembled on a scaffold of decondensed chromatin fibers in a process termed NETosis, which has been proposed to protect tumor cells from T cell- or natural killer (NK) cell-mediated toxicity (Teijeira et al., 2020). This should be further studied by developing a specific antibody panel to visualize and measure NET formation in tissue. Other upregulated antimicrobial genes such as the chemokine CXCL3 could contribute to recruitment of neutrophils at the ITF. Altogether, it is tempting to speculate that impaired antimicrobial humoral response could contribute to local or distant spread of uterine uLMS.

Epigenomic analysis showed a generally similar methylation pattern in the ITF of uADC and uLMS. However, when we focused our analysis on CGI promoters, we observed a different methylation profile in the ITF of the two tumor types. Interestingly, part of these differentially methylated genes was involved in biological processes related to cell adhesion and ECM organization. Of note, both biological pathways play a major role in invasion and migration of several types of tumors, including uterine neoplasms (Abal et al., 2007; Yadav et al., 2020), suggesting that epigenetic disruption of these pathways could influence the metastatic properties of uADC and uLMS. In addition, combining transcriptomic and epigenomic analysis, we observed an association between the methylation status of CGI promoters and expression in a set of 20 genes, implying epigenetic regulation of these genes in ITF. Importantly, this 20-gene epigenetic signature was able to clearly differentiate between ITF of uADC and uLMS. One of these genes was ITGB3, an integrin involved in cell adhesion and ECM whose deregulation is associated with cancer metastasis (Langsenlehner et al., 2006; Hu et al., 2018). Another epigenetically regulated gene identified in this signature was CXCL16, a chemokine-derived peptide implicated in antimicrobial and antitumoral response (Valdivia-Silva et al., 2015). Cancer patients with high levels of CXCL16 expression have shown more elevated levels of CD4(+) and CD8(+) tumor-infiltrating lymphocytes and NK, leading to improved disease prognosis (Hojo et al., 2007; Valdivia-Silva et al., 2015). However, this chemokine can also induce invasion and metastasis via several mechanisms, such as the recruitment of mesenchymal stem cells into the tumor (Jung et al., 2013). In addition, CXCL16 contributes to ectopic endometrial stromal cell migration and invasion (Peng et al., 2019). We also detected other epigenetically regulated genes in this signature, including CD70, IRAK, IL20RA, THBD, TACSTD2, and MST1R, with implications related to the tumor immune microenvironment (Jain et al., 2014; Rossi et al., 2014; Babicky et al., 2019; Gu et al., 2020; Kumar et al., 2020; Gao et al., 2021). In our study, TACSTD2 was overexpressed and hypomethylated in the ITF of uADC in contrast to uLMS. Interestingly, other studies have found highly expressed TACSTD2 in aggressive endometrial carcinomas associated with tumor invasion (Bignotti et al., 2011), and it has been suggested as an attractive target for cancer immunotherapy in biologically aggressive endometrial carcinomas (Bignotti et al., 2012). Collectively, these data indicate that although the ITFs of uADC and uLMS have a similar global methylation status, there are also some significant epigenetic differences that could influence the invasive, migration, and tumor microenvironment properties of the two types of uterine neoplasms.

In conclusion, our results demonstrate that uADC and uLMS share several morphometric and molecular features at the interface between tumor cells and myometrium, but also an intertumoral heterogeneity. There was a high degree of similarity between tumor and ITF in reticulin scaffolding, macrophage infiltration, and global DNA methylation pattern. However, uLMS had fewer T and B lymphocytes; additionally, neutrophil degranulation releasing antimicrobial peptides was observed in uADCs, but not in uLMS. Furthermore, gene expression profiling and DNA methylation identified a 20-gene epigenetic signature characteristic of ITF. It seems that the two tumors exhibit certain remarkable similarities when invading the myometrium despite being different neoplasms with profound morphologic and molecular differences, and originating from different precursor cells. These findings open up the possibility of investigating common therapeutic strategies based on these similarities in the ITF microenvironment. The results reported in this article have various potential clinical implications. The characteristics of the tumor microenvironment and immune surveillance in these two types of tumor suggest that targeting non-cancer cells, or mediators of their communication, may be efficient across different tumor types and could also complement other therapies. Moreover, tumor microenvironment features may provide insight into the mechanisms underlying the failure of previously effective anticancer drugs.

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 below: Methylation and expression profiling have been deposited at the NCBI GEO repository with accession numbers GSE171142 and GSE172034 respectively.

The studies involving human participants were reviewed and approved by the Comité Coordinador de Ética de la Investigación Biomédica de Andalucía: Deciphering the site-specific tumor microenvironment of advanced uterine tumors (Biobank code: S1800086) and Comité de Ética de la Investigación with the codes CEIC-1892, CEIC-2083, and CEIC-1858. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

RN and XM-G contributed to conception and design of the study. SS wrote the first draft of the manuscript. SS, NE, CA, JD-M, ÁD-L, EA, XM-G, and RN wrote sections of the manuscript. BD, RL, SR, EA, and XM-G provided the clinicopathological data. NE provided the collaborative consortium with microdissected samples. SS and RN designed and performed the morphometric characterization of the reticulin fibers. CA and DG performed the multiplex staining and analysis. JD-M, CS-A, and EA evaluated the transcriptomic profile. ÁD-L and MJ performed the genome-wide methylation analysis. All authors contributed to manuscript revision, read, and approved the submitted version.

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.