Dissecting the immune landscape of tumor draining lymph nodes in melanoma with high-plex spatially resolved protein detection

Abstract

Background: In melanoma patients, microscopic tumor in the sentinel lymph-node biopsy (SLN) increases the risk of distant metastases, but the transition from tumor in the SLN to metastatic disease remains poorly understood.

Methods: Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g., tumor) or by fluorescent cell subset markers (e.g., CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune-related proteins were collected and normalized to account for system variation and ROI area.

Results: Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years.

Conclusion: We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

Keywords: Melanoma; Tumor draining lymp nodes.

Fig. 1

Analysis of sentinel lymph node from patient 3. a Low-power H&E. b High-power H&E of outlined tumor region from (a). c Low-power fluorescent staining CD11c (green), CD20 (yellow), CD3 (red), DNA (blue), regions 1, 2, 3, and 6 outlined in white are tumor within the sentinel lymph node, while 4 and 5 are histologically normal appearing sections of the lymph node. d High power of region 6 from (c). e High-power region 2 from c and orange circle highlights CD11c (green) cells. f High-power region 5 from (c). g Same region as f with only CD11c cells visible. h High-power region 4 from (c). i Same as region h with only CD11c cells visible

Fig. 2

Comparison of tumor regions of the sentinel lymph node (SLN) for patients 1, 2, 3. Fluorescent antibodies were used to examine CD11c (green), CD20 (yellow), CD3 (red), and DNA (blue). a High-power SLN tumor region for patient 1. b Same region as a with only CD11c cells visible. c Same region as a, b with CD11c cells outlined in white. Protein expression was run on these outlined cells. d High-power SLN tumor region for patient 2. e Same region as D with only CD11c cells visible. f Same region as d, e with CD11c cells outlined in white. Protein expression was run on these outlined cells. g High-power SLN tumor region for patient 3. h Same region as g with only CD11c cells visible. i Same region as g, h with CD11c cells outlined in white. Protein expression was run on these outlined cells

Fig. 3

Protein expression on CD11c cells. a Percent change in signal:noise protein expression on non-tumor CD11c cells to tumor CD11c in SLN of patients 1, 2, and 3 sorted by highest percent change. Positive values indicate higher protein expression on CD11c cells in SLN tumor to CD11c SLN non-tumor. *indicates values higher than the upper limit presented in graph. The percent change for MART-1 in patient 1 was 502%, patient 2 was 1341%, and patient 3 was 2451%. The percent change for S100B in patient 2 was 2596% and patient 3 was 1218%. b Signal:noise ratio of selected proteins on CD11c cells only in SLN tumor regions and non-tumor regions for patients 1, 2, and 3 and CD11c from patient 4 who had no tumor in the SLN

Fig. 4

Analysis of patient 1 primary tumor and sentinel lymph node. Patient 1 primary tumor (panels a, b, c, d) and sentinel lymph node (panels E, F, G, H). a H&E low-power primary tumor. b High power of region that is outlined in (a). c Low-power primary tumor with fluorescent staining for CD11c (green), CD20 (yellow), and CD3 (red), DNA (blue). d High-power region 1 of primary tumor that is outlined in white in (c). e Lower power SLN, region 1 outlined in white is microscopic tumor in the SLN. f High-power region 1 from (e). g Low-power H&E of SLN. h High power of region outlined in white in (g)

Fig. 5

Analysis of patient 2 primary tumor and sentinel lymph node. Patient 2 primary tumor (panels a, b, c, d) and sentinel lymph node (panels e, f, g, h). a H&E low-power primary tumor. b High-power primary tumor region outlined in white in (a). c Low-power view of SLN with fluorescent staining for CD11c (green), CD20 (yellow), CD3 (red), and DNA (blue). d High-power region of primary tumor outlined in white in (c). e Lower power SLN, the region outlined in white is microscopic tumor in the SLN. f High power of the tumor region outlined in white in (e). g Low-power H&E of SLN. h High power of tumor region outlined in white in (g)

Fig. 6

Percent change in signal:noise protein expression of tumor in the sentinel lymph node to the primary tumor. Positive values indicate higher protein expression in the primary tumor. *indicates values higher than the upper limit presented in graph. The percent change for fibronectin in patient 2 was 1773%. The percent change for CD45RO was 175 for patient 1