Fig 2: Macrophage polarization in the lymph node sinus depending on the L-status of the primary tumor. a) The figure shows the ratio between the CD11c cell count and the CD68 cell count in the lymph node sinus (LN sinus) as indicator of M1 polarization. Tumor draining lymph nodes free of metastasis have been examined. P-values generated by the ANOVA-test are indicated. A significantly decreased CD11c/CD68 ratio in the lymph node sinus can be found in cases with lymph vessel infiltration at the primary tumor site (L1) compared to L0 cases. b) The figure shows the ratio between the CD163 cell count and the CD11c cell count in the lymph node sinus (LN sinus) as indicator of M2 polarization. Tumor draining lymph nodes free of metastasis have been examined. P-values generated by the ANOVA-test are indicated. A significantly increased CD163/CD11c ratio in the lymph node sinus can be found in in cases with lymph vessel infiltration at the primary tumor site (L1) compared to L0 cases. c) The figure shows the ratio between the MRC1 cell count and the CD11c cell count in the lymph node sinus (LN sinus) as indicator of M2 polarization. Tumor draining lymph nodes free of metastasis have been examined. P-values generated by the ANOVA-test are indicated. A significantly increased MRC1/CD11c ratio in the lymph node sinus can be found in in cases with lymph vessel infiltration at the primary tumor site (L1) compared to L0 cases.

Fig 3: Typical expression pattern of the macrophage markers CD68, CD11c, CD163 and MRC1 in a lymph node. Exemplary fields of view (original magnification 40x) showing the typical expression pattern of the stained macrophage markers CD68, CD11c, CD163 and MRC1 in lymph node specimens. a) CD68: The marker shows a cytoplasmatic staining. The shape of the stained cells includes predominantly round cells, but some cells also show a spindle-shape. CD68 is expressed in the follicles, in the interfollicular zone and in the sinus. Besides a distinct staining of the macrophages, in lymph node metastasis specimens a pale staining of tumor cells was also detectable. b) CD11c: The marker shows a cytoplasmatic expression pattern with an accentuation of the plasma membrane. The shape of the stained cells includes predominantly round cells. CD11c expressing cells were mainly found in the follicles and in the interfollicular zone. c) CD163: The marker shows a cytoplasmatic expression pattern with an accentuation of the plasma membrane. The shape of the stained cells includes predominantly spindle-shaped cells. CD163 expression can predominantly be found in the lymph node sinus. The follicles are largely missing CD163 expressing cells. d) MRC1: The marker shows a cytoplasmatic expression pattern with an accentuation of the plasma membrane. The shape of the stained cells includes predominantly spindle-shaped cells. MRC1 expression can predominantly be found in the lymph node sinus. The follicles are largely missing MRC1 expressing cells.

Fig 4: Macrophage cell count (cells/mm2) in tumor free and metastatic lymph nodes depending on the L-status of the primary tumor. The figure shows the median macrophage cell count (cells/mm2 specimen area) in the lymph node sinus (LN sinus) depending on the lymph vessel infiltration status of the primary tumor (L0 vs. L1). Both groups of tumor draining lymph nodes have been examined (lymph nodes free of metastasis and metastatic lymph nodes). P-values generated by the ANOVA-test are indicated. a) CD68 cell count: A significantly increased count can be observed in the L1 cases. b) CD11c cell count: A significantly decreased count can be observed in the L1 cases. c) CD163 cell count: A significantly increased count can be observed in the L1 cases. d) MRC1 cell count: A significantly increased count can be observed in the L1 cases.

Fig 5: Distribution of Galectin 3 and macrophage marker expressing cells in lymph nodes. The figure displays the typical expression pattern of Galectin 3 positive cells (a) compared to the established macrophage markers CD68, CD11c, CD163 and MRC1. CD68 (b) is the best established pan-macrophage marker for M1- and M2-polarized macrophages. CD11c (c) is a marker for M1-polarized macrophages. CD163 (d) and MRC1 (e) are M2 macrophage markers. Corresponding virtual microscope images (20× magnification) of a tumor-free cervical lymph node of a oscc patient are shown