Understanding each individual cell type in cancer progression is paramount to the development of new treatments and identification of appropriate therapies. To shed light on how specific immune cells impact tumor development, researchers often draw on robust single-cell analysis.
Recently, a team of scientists at The University of Texas MD Anderson Cancer Center used this process to create a single-cell map of tumor-infiltrating B cells (TIBs) and plasma cells in early-stage lung adenocarcinoma (LUAD). According to the research, LUAD remains the most frequently diagnosed histologic subtype of lung cancer and accounts for most cancer deaths related to smoking. This article explores the study's findings and how the results may help to better understand the transcriptional, clonotypic states, and phenotypes of TIBs.
To date, the researchers expressed that TIBs have been poorly characterized and relatively understudied, and the analysis of the immunobiology of LUADs and other solid tumors has been largely T cell-focused. This could be a missed opportunity, as growth factors continue to impact the likelihood of recurrence. This is despite lung cancer being detected earlier in patients through enhanced screening measures. According to the National Cancer Institute, regardless of cancer stage, grade, or type of lung cancer, patients were more likely to have distant metastasis than local recurrence. In Stage IA and IB disease, about one-third of patients had a recurrence.
"The design of this study is in the early-stage lung cancer and uses surgical resection samples. It's important to understand the roles of different B-cell and plasma cell subpopulations in disease progression and metastasis moving forward," shared co-corresponding author Linghua Wang, MD, PhD, Associate Professor of Genomic Medicine at MD Anderson Cancer Center. This study was jointly supervised by Humam Kadara, PhD, Associate Professor of Translational Molecular Pathology at MD Anderson Cancer Center.
Originally published in the journal Cancer Discovery, the team's research involved working to create a comprehensive single-cell atlas to better understand the single-cell and spatial landscape of TIBs in early-stage LUADs and interrogate how the tumor microenvironment phenotype of LUAD is modulated by TIBs (2022; doi: 10.1158/2159-8290.CD-21-1658). Thus, the atlas explored their transcriptional states, differentiation and maturation status, geospatial characteristics, and clonotypic properties. It also explored the cellular interactions and colocalization patterns with other TME cell populations.
Additionally, the authors correlated TIB characteristics with major clinicopathologic features, including smoking, driver gene mutation, and tumor stage. They also assessed their clinical significance, such as patient survival and response to immunotherapy. The team wanted to further analyze the early interactions in space between cancer cells and the phenotypes of B cells and plasma cells to potentially gain greater insight into how they impact the tumor microenvironment of LUAD. Ultimately, their goal was to use this information to determine if there is a better way to trigger an anti-tumor immune response to block cancer cells from ever advancing. To do so, they employed several methods.
These included the multiregional sampling of surgically resected LUADs and matched normal lung tissues, as well as paired single-cell RNA and immune repertoire sequencing. The team of researchers also used state-of-the-art bioinformatics analyses and performed validation at various levels, such as using IHC staining, MIF assay, NanoString GeoMX DSP, and cutting-edge spatial transcriptomics.
To assess phenotypic relationships and examine co-occurrence patterns (both positive and negative) between TIB cell subsets and various other immune and stromal cell populations in the TME, they applied Spearman correlation analysis. Then, to compare the fractions of different B cells and plasma cells (PC) and their subsets across tumor and spatially defined normal tissues, the "Mann-Whitney test and paired t-test were applied" as indicated, according to the research.
In general, "all statistical significance testing in this study was two-sided, and results were considered statistically significant at P values or FDR q-values <0.05." According to the research, "defaulted P<2 x 10-16 reported in R v3.6.0 was used when the P value was too small to illustrate," (Cancer Discov 2022; doi: 10.1158/2159-8290.CD-21-1658).
"B cells and plasma cells in the tumor have multifaceted roles, the plasma cells can secrete antibodies and kill cancer cells, and TIBs have additional ways to kill cancer cells. However, some TIB subsets are immunosuppressive. As we have a better understanding of the TIBs and immunobiology of lung cancer, I think we can develop better strategies in the future to effectively modulate those unfavorable subpopulations in the tumor microenvironments," Wang explained. "That may also help us identify new biomarkers for a better patient stratification, develop B-cell targeted therapy, or [use a] combination of therapy with the T cells so that more cancer patients can benefit from cancer immunotherapy."
Ultimately, this study followed previous research also conducted by The University of Texas MD Anderson Cancer Center that has been cited over 800 times (Nature 2020; https://doi.org/10.1038/s41586-019-1906-8). The prior paper found that the "likelihood of a patient responding to immune checkpoint blockade may depend on B cells in the tumor, located within specialized immune-cell clusters known as tertiary lymphoid structures (TLS)."
As such, checkpoint inhibitors offer the potential for long-term survival to patients across many cancer types, and it was thought that interaction between components of the immune system could offer preventative checkpoint blockades.
"Our teams at MD Anderson discovered that B-cell signal is important and associated with immune therapy response in patients with melanoma and sarcoma and was associated with better survival," Wang shared. "Those were important landmark studies in the field as we noticed there was a remarkably increased abundance of TIBs in lung adenocarcinoma in another study published by our team in Cancer Discovery last year, the study was jointly led by Dr. Kadara and myself. That's why we decided to look further into this."
Study Details
To initiate this follow-up B-cell research, Wang, Kadara, and their co-authors thereby performed single-cell analysis on the tumors of 16 patients with early-stage LUADs (I-IIIA) and 47 matching multi-region normal tissues. Further, single-cell RNA sequencing was performed on roughly 50,000 unique B cells and plasma cells to analyze their gene expression profiles, as was single-cell B-cell receptor sequencing on more than 70,000 cells.
To validate this finding and further examine the spatial distribution of TIBs, the researchers shared that they had performed "multiplexed fluorescence (mIF) with a panel of eight markers on 20 available tissues from five of these 16 patients." They reported that, of all the tumor samples collected from patients, the lymphoid aggregates (LAs) were detected in the tumor compartment and stroma. LAs were also present in the nontumoral stromal compartment of two patients, and "the total intensities of B cells were significantly higher in tumors compared with their matched normal tissues," the authors reported.
A key takeaway from this methodology was that most B cells and plasma cells were found to be recruited to sites with high levels of CXCL13. This signaling molecule's levels were shown to increase as tumors progressed from precancerous lesions to invasive lung cancer. Through the assessment of single-cell data together with spatial information from the tumors, the researchers saw that the varied landscape of B cells and plasma cells in the tumor influenced patient treatment responses in early-stage lung cancers and overall outcomes. According to the research, enrichment of plasma cells in the tumor was associated "most strongly with improved survival and responses to anti-PD-1/PD-L1 immune checkpoint inhibitors."
By the study's conclusion, the researchers were able to identify 12 TIB and plasma cell subsets. In addition to memory B cells and plasma cells, other differentiated states were identified as being highly enriched in the tumors relative to adjacent normal tissue. The single- cell profiling of B-lineage cells in the tumor matched the multiregional normal lung tissues in 63 samples from the 16 patients with early-stage LUADs.
These findings noted that "prominent increase in the fractions of PCs in smokers with more long-lived PCs are in agreement with the observation of the decreased fractions of PCs and lower SHM rates." This was found to be the case in LUADs "harboring EGFR mutations," which the authors expressed is commonly seen in nonsmokers. According to the research, the results also indicate a "superior role of PCs in smoking-related LUADs."
Another pivotal takeaway from the analyses was the highly abundant IgA+ PCs in LUADs "whereby IgA+ PCs strongly co-occurred with activated and proliferative Tregs, CD8+ Tex, CAFs, and macrophages and negatively correlated with CTLs." This was reportedly consistent with "multiple lines of evidence supporting the immunosuppressive and protumorigenic role of IgA+ PCs (30)." However, the researchers stressed that "further functional studies are needed to determine their exact roles in LUAD pathogenesis."
"Most studies treat B cells and plasma cells each as a homogenous population. This study tried to leverage their power as we had more than 50,000 cells that gave us the power to look into the heterogeneity in their cell states at various levels," Wang explained. "With unbiased clustering analysis, we identified distinct B-cell subsets, as we're also able to infer the immunoglobulin isotypes of plasma cells based on their BCR sequences. This also led us to better understand the heterogeneity among this population."
Upon publication, these results have truly highlighted the heterogeneous nature of B cells and plasma cells, along with their interaction with other tumor microenvironment components. While the studying of the single-cell data together with spatial information from the tumors yielded this insight, Wang noted that more functional studies are needed to clarify the precise roles of B cells and plasma cells in early lung tumor progression.
"There are still too many unknowns. Moving forward, TIBs has a bright future and could be a promising target in terms of modulating the tumor microenvironment in lung cancer and possibly also in other cancer types. Currently, we do not know what exactly they're doing in the microenvironment. We saw different TIB populations in the tumor microenvironment, but we do not know if they are killing tumors or fighting pathogens or just bystanders," Wang explained. "We have to figure out their antigen specificity, roles, and dynamics."
She added that this study provided a snapshot of TIBs in LUAD. Wang believes the next logical step would be to design longitudinal and spatial studies to look at their dynamics in abundance, composition, and phenotypes during the continuum of LUAD from pre-cancerous lesions to early-stage cancers and then to advanced cancers, and investigate TIBs in the context of immunotherapy, combined with well-designed pre-clinical studies.
"That will lead to a better understanding of their roles and how to develop strategies to modulate those populations and translate into clinic to improve patient care," she concluded.
Lindsey Nolen is a contributing writer.
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