Case report: Understanding the impact of persistent tissue-localization of SARS-CoV-2 on immune response activity via spatial transcriptomic analysis of two cancer patients with COVID-19 co-morbidity

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected half a billion people, including vulnerable populations such as cancer patients. While increasing evidence supports the persistence of SARS-CoV-2 months after a negative nasopharyngeal swab test, the effects on long-term immune memory and cancer treatment are unclear. In this report, we examined post-COVID-19 tissue-localized immune responses in a hepatocellular carcinoma (HCC) patient and a colorectal cancer (CRC) patient. Using spatial whole-transcriptomic analysis, we demonstrated spatial profiles consistent with a lymphocyte-associated SARS-CoV-2 response (based on two public COVID-19 gene sets) in the tumors and adjacent normal tissues, despite intra-tumor heterogeneity. The use of RNAscope and multiplex immunohistochemistry revealed that the spatial localization of B cells was significantly associated with lymphocyte-associated SARS-CoV-2 responses within the spatial transcriptomic (ST) niches showing the highest levels of virus. Furthermore, single-cell RNA sequencing data obtained from previous (CRC) or new (HCC) ex vivo stimulation experiments showed that patient-specific SARS-CoV-2 memory B cells were the main contributors to this positive association. Finally, we evaluated the spatial associations between SARS-CoV-2-induced immunological effects and immunotherapy-related anti-tumor immune responses. Immuno-predictive scores (IMPRES) revealed consistent positive spatial correlations between T cells/cytotoxic lymphocytes and the predicted immune checkpoint blockade (ICB) response, particularly in the HCC tissues. However, the positive spatial correlation between B cells and IMPRES score was restricted to the high-virus ST niche. In addition, tumor immune dysfunction and exclusion (TIDE) analysis revealed marked T cell dysfunction and inflammation, alongside low T cell exclusion and M2 tumor-associated macrophage infiltration. Our results provide in situ evidence of SARS-CoV-2-generated persistent immunological memory, which could not only provide tissue protection against reinfection but may also modulate the tumor microenvironment, favoring ICB responsiveness. As the number of cancer patients with COVID-19 comorbidity continues to rise, improved understanding of the long-term immune response induced by SARS-CoV-2 and its impact on cancer treatment is much needed.

Keywords: SARS-CoV-2; case report; immunotherapy; intra-tumor heterogeneity; spatial transcriptomics; tissue-localized immunity.

Figure 1

Spatial enrichment of COVID immune response signatures in biopsied HCC and CRC tissues from COVID-19-recovered patients. (A–P) Visium-based (10× Genomics) spatial transcriptomics-generated tissue heat maps show the spatial localization of COVID immune responses in HCC (A, E, I, M) and CRC (C, G, K, O) tumors and their adjacent normal tissues (B, F, J, N, D, H, L, P, respectively). Analysis was based on lymphocyte-associated COVID immune response gene signatures (Lee et al. (15), A–D; and Ren et al. (16), E–P): CD8⁺ T/NK cells-associated (A–D), B cell-associated (E–H), T cell-associated (I–L), and NK cell-associated (M–P).

Figure 2

Distribution of SARS-CoV-2 spike protein (SP) and immune cell types in the spatial transcriptomic (ST) niches of HCC and CRC biopsies from COVID-19-recovered patients. (A–D) From left to right: HCC, HCC-adjacent normal, CRC, and CRC-adjacent normal tissues; ST niches (clusters 1–4) were determined using the spatially-aware BayesSpace (BS) transcriptomic clustering method. (E–L) RNAscope-detected SARS-CoV-2 SP was quantified in regions of interest (marked as boxes) in the Visium-defined ST niches (E–H); see Supplementary Table 2 for raw data and assignment of ROIs to ST niches. Bar charts (I–L) show the relative SP counts in the ST niches; SP counts in ROIs assigned to the same ST niche were averaged. Areas with poor staining quality (F) and smooth muscle and collagen (H, L) were omitted. (M) Distribution of immune cell abundance (estimated by a deconvolution-based microenvironment cell population-counter) in ST niches. The orange stars indicate viral-high regions (ST niches with the highest SARS-CoV-2 SP counts).

Figure 3

Spatial colocalization of immune cell types and COVID immune response. (A–D) Immune cell types (determined by a deconvolution-based microenvironment cell population-counter) and COVID immune responses (quantified as enrichment scores for the COVID response-associated gene signatures reported in Lee et al. (15) within the viral-high regions in HCC (A) and CRC (C) tumors and the corresponding adjacent normal tissues (B, D, respectively). Lymphocyte and myeloid-associated COVID responses are represented by triangles and circles, respectively. The vertical green dashed line delineates log-fold change (FC) = 0 (log-FC > 0 for cell types with higher COVID immune scores in Visium spots where the immune cell type was detected than in spots where it was not detected). The horizontal green dashed line represents P = 0.05; above the line, P < 0.05. Red text and annotations represent P < 0.005. P-values were computed using the Kruskal-Wallis test. CTLs, cytotoxic lymphocytes; DCs, dendritic cells.

Figure 4

Spatial associations of immune cell types and immuno-predictive scores (IMPRES) in individual spatial transcriptomic niches. From left to right, BayesSpace (BS) clusters 1–4 in HCC, HCC-adjacent normal tissue, CRC, and CRC-adjacent normal tissue are shown. Each BS cluster was stratified by the presence of the immune cell of interest (determined by a deconvolution-based microenvironment cell population-counter) as follows: B lineage, T cells, CD8⁺ T cells, cytotoxic lymphocytes, NK cells, myeloid dendritic cells, monocytic lineage, and neutrophils (from top to bottom). Orange stars indicate viral-high regions (transcriptomic niches with the highest SARS-CoV-2 spike protein/nucleocapsid protein counts); red arrows indicate higher IMPRES scores in Visium spots analyzed for B lineage cells, T cells, and cytotoxic lymphocytes within the viral-high regions.