{
  "abstract": "Background Immune checkpoint inhibitors have significantly improved survival for melanoma patients, yet 40-60% remain unresponsive to treatment. Identifying pathways of resistance and developing novel therapies are crucial steps for improving melanoma outcomes. Tumor-draining lymph nodes (TDLNs) act as an extension of the tumor microenvironment, influencing local and systemic immunity and responses to immunotherapies. Within lymph nodes (LNs), fibroblastic reticular cells (FRCs) are critical orchestrators of immune responses, and they can potentially influence melanoma anti-tumor immunity.Methods To analyze FRCs in human melanoma TDLNs, we performed multiplexed immunofluorescence on archival TDLN tissues. Our cohort included samples from six healthy controls and 21 melanoma patients (11 Stage II, 10 Stage III, including metastatic and non-metastatic TDLNs). Tissues were stained with PDPN and CD31 antibodies to identify FRCs (PDPN+CD31–), excluding other stromal cells. To further characterize melanoma TDLN-FRCs, we also conducted ex vivo morphology, phenotypic, and transcriptomic analysis of primary human FRCs freshly isolated from Stage IIIC melanoma patients undergoing lymphadenectomy, comparing them to FRCs from healthy individuals.Results Image analysis of LN tissues revealed significant remodeling of the FRC network in melanoma TDLNs. This altered network co-expressed alphaSMA and featured larger spaces between reticular branches, indicating a loosened FRC network, typically observed during immune responses or in pathological conditions. Interestingly, melanoma LNs are characterized by gradients of FRC network remodeling that correlated with the clinical stage and TDLN metastatic involvement.To assess whether melanoma directly influenced FRC status, we co-cultured primary human FRCs with melanoma cell lines or conditioned them with melanoma supernatant. Morphological and phenotypical analysis of healthy human FRCs revealed that melanoma exposure induced FRC activation. This activation was further confirmed by cell shape assessment of FRCs isolated directly from stage IIIC melanoma patients, showing consistent stretching compared to healthy counterparts.To gain deeper insights into melanoma-induced FRC remodeling, we performed RNAseq on freshly isolated FRCs from melanoma TDLNs (5 Stage III melanomas). Our analysis revealed significant enrichment in pathways associated with cell proliferation, metabolism, and extracellular matrix remodeling in melanoma FRCs compared to healthy controls. Additionally, we observed changes in immunomodulatory genes, particularly those linked to MHC and chemokines.Conclusions Our results indicate that melanoma significantly remodels FRCs in TDLNs, and potentially impacting on the immunomodulatory role of LN fibroblasts. Current analyses are focusing on the dysregulated immunological signatures in melanoma-FRCs to understand if and how they influence anti-tumor immunity.Ethics Approval All patient samples were obtained with written informed consent, in accordance with the Declaration of Helsinki, and their research use was approved by the local Ethics Committee of Istituto Tumori ‘Giovanni Paolo II’ of Bari (CE prot. n. 10425 del 28.04.23)",
  "authors": [
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Benedetta Apollonio"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Chiara Bungaro"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Eustachio Ruggeri"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Domenica Lardo"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Aurelio Costa"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Andrea Armenio"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Benedetta Di Venosa"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Angela Ricco"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Ivana De Risi"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Ileana De Roma"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Sabino Strippoli"
    },
    {
      "affiliations": [
        "IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy"
      ],
      "name": "Michele Guida"
    }
  ],
  "title": "1212 Immunomodulation beyond the tumor microenvironment: FRC reprogramming in melanoma-draining lymph nodes",
  "uid": "c2d4467a-6399-5630-9335-482db8c1e8b5"
}