Pancreatic cancer (PDAC) remains a deadly disease and is notoriously challenging to treat with a 5-year survival rate of 10% (1). While the only curative treatment is complete surgical resection, fewer than 20% of patients are eligible due to advanced stages of disease, making medical therapies the mainstay for most PDAC patients. Unfortunately, while PDAC initially responds well to the standard combination therapies of FOLFIRINOX or gemcitabine/nab-paclitaxel, most patients progress due to chemoresistance, leading to poor outcomes (2). As the mechanisms of tumor progression and chemoresistance are multifactorial and poorly understood, there is an unmet need for the development of better treatment strategies. Growing evidence demonstrates that the tumor microenvironment (TME) is a vital component in the pathogenesis of PDAC and plays an essential role in tumor progression, invasion, and therapeutic resistance (3). Desmoplastic stroma comprises up to 80% of total tumor volume and largely consists of immune cells, fibroblasts, and acellular collagens (4). In particular, the accumulation of myeloid cells in the TME drives immune suppression (5–7). Previous studies have shown that targeting the myeloid compartment within PDAC tumors in murine models led to increased cytotoxic T cell activity, decreased regulatory T cell activity, shrinkage of tumors, and improved survival (5–7); however, clinical trials targeting myeloid cells have failed or only partially recapitulate results from preclinical models in a subset of patients (8, 9). This highlights the lack of fidelity in using preclinical murine models for human PDAC.

Strategies to molecularly profile human pancreatic tumors are thus crucial to help unravel the complexities of human disease. Recently, single-cell RNA sequencing (scRNA-seq) has been shown to provide the analytical power to define cell-specific molecular signatures and map out the interactions of cell types within the TME (10–15) ( Figure 1 ). This analytical strategy is capable of characterizing cell types and states in an unbiased manner, and is key to elucidating the behavior of myeloid cells within the PDAC TME. Ultimately, harnessing the power of scRNA-seq technology can help unravel the intricacies of these cell-cell interactions in human PDAC and lead to the development of novel therapies targeting the microenvironment to improve outcomes for this dismal disease. In this review we highlight the latest progress made in classifying myeloid cell compartment in PDAC by unbiased single-cell analysis ( Figure 2 ).

Within the immune compartment of the PDAC microenvironment, myeloid cells have been shown to be key regulators of immunosuppression and strong correlators to poor clinical outcomes (16, 17).They are abundant in the TME by way of myeloid-promoting cytokines such as CSF-1 and CCL-2 (18, 19). Perhaps their most well-known role in PDAC is their ability to mitigate anti-tumor effector T cell function through the release of cytokines that are immunosuppressive and in turn recruit other cells known to further dampen cytotoxic immune responses, such as T regulatory cells (20). Myeloid cells also mediate the expression of immune checkpoint ligands on tumor cells as another mechanism of immune evasion (21).

Myeloid cells are also known to play other roles that are independent of T cell responses. Tumor-infiltration of these cells is critical for PDAC initiation, as they directly promote the formation and maintenance of preneoplastic lesions through factors including EGF ligand and PDGF in murine models of PDAC (22, 23).

Interestingly, myeloid cells have been shown to directly enhance chemoresistance in PDAC tumor cells in vitro using indirect co-culture assays, implicating soluble factors as mediators (24, 25). It has been previously shown that conditioned media from tumor-educated bone marrow-derived macrophages confers chemoresistance to gemcitabine in vitro, specifically through pyrimidine release in myeloid cells (25).

Studies have also suggested that myeloid cells play a vital role in the pre-metastatic niche as a precursor colonizer to metastatic sites that allow for a favorable environment for tumor cell seeding and growth (26, 27). Recently, a new role of myeloid cells was uncovered in mouse models linking myeloid cell invasion into the central nervous system leading to cachexia symptoms in PDAC (28).

While these studies provide information about the behavior of myeloid cells and their response to numerous environmental stimuli to promote PDAC pathogenesis, the bulk of these studies were performed in preclinical murine models, with limited correlation in human studies. Recent single cell studies on human tumor tissue have allowed for a better understanding of the transcriptional diversity and putative function of myeloid cells in human disease.

Previously, characterization of the myeloid compartment within TME in human studies was limited to immunostaining and flow cytometric techniques, while more in-depth study of transcriptional networks involved in the myeloid compartment of PDAC tumors could only be determined through deconvolution methods (29) (See Table 1 ). However, with the advent of multidimensional single-cell and spatial techniques, we now know that the myeloid compartment in PDAC has a complex heterogeneity. It is important to note that there is a diverse array of different transcriptomic patterns of myeloid cells across different cancer types, and care should be taken before generalizations are made regarding the myeloid transcriptome of PDAC, which varies from other solid tumor types (30). Table 2 summarizes significant contribution to human single cell sequencing on PDAC to date.

Single cell transcriptomic technology has shed much-needed light on the heterogeneity and function of the myeloid compartment in human PDAC. While pre-clinical murine models have dominated the field in the study of the tumor immune microenvironment, results from these studies have led to an oversimplification of the myeloid cell types and have resulted in identifying targets that thus far have had mixed patient outcomes (8, 9, 53). The patient heterogeneity in drug response of these clinical trials is supported by single-cell studies, which highlight the inter-patient heterogeneity of the myeloid compartment. In many of the trials reviewed above, a small subset of patients had some response to the given immunotherapeutic strategy, suggesting that a precision medicine-based platform is needed which can match therapy to each tumor’s microenvironmental characteristics. While there is no such tool in place to tailor these therapies, single-cell transcriptomics bring a promising avenue for both biomarker and therapeutic discovery.

One caveat to note is that the technique of single cell transcriptomics is not without its own flaws, which include variation in tissue acquisition and processing, read “dropout”, and, unfortunately, false discoveries (60, 61). Therefore, validation of gene expression through complementary techniques such as multiplex immunofluorescence or mass cytometry is necessary ( Table 1 ). Furthermore, putative interactions and identified signaling networks should be investigated with further functional studies using in vitro or in vivo systems.

Another limitation to single cell RNA sequencing is that spatial data is not preserved, and validating targets via immunostaining can be laborious. Indeed, work using multiplex immunofluoresence has shown that immune cell localization of myeloid cells within the tumor had important clinical significance in PDAC patients (62). Recent developments in spatial transcriptomics and multiplex staining can add a crucial dimension to identifying cell subtypes in the TME and validating putative crosstalk between cells (16, 63). Alternatively, machine learning has been leveraged with multiplexed immunofluorescence and whole-slide imaging for tissue segmentation and classification (64, 65). Recent bioinformatics pipelines are actively working to integrate these multi-dimensional datasets for a seamless approach and yield new insights on myeloid cells in the TME (66–68).

In conclusion, PDAC remains a deadly disease with an urgent need to find new and better therapies. Targeting the myeloid compartment of the TME is a promising avenue to pursue; although given the complexities of these cells shown by single cell studies, single-agent immunotherapy is likely not sufficient and combinatorial approaches may be required. One exciting avenue to apply single-cell transcriptomics is through the study of tumor tissue longitudinally throughout the course of disease and therapy treatment, as myeloid cells have been shown in preclinical studies to play a major role in the development of chemoresistance (24, 25). Leveraging this technique to comprehensively study the immune microenvironment in the treatment-naïve and post-treatment states may provide new insights to the role of the TME in the development of chemoresistance and ultimately identify new pathways to target in this dismal disease.

All authors participated in writing the manuscript, with EC taking the lead in organizing and compiling the final document. PK and EC illustrated Figures 1 , 2 . All authors contributed to the article and approved the submitted version.

This work was supported by the American College of Gastroenterology Career Development Award and the VA BLR & D Career Development Award to EC; T32-AI007413 to PK.

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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