Transmembrane chemokines act as receptors in a novel mechanism termed inverse signaling

Essential revisions:

Overall, the studies are well described and performed. Clearly, the results are novel and will likely be of interest. All three reviewers agreed on the interest in the studies and the potential significance of the findings. The biggest criticism is that no parallel in vivo data is provided. This is somewhat limiting, especially as the authors discuss the results in the context of tumor biology. Ideally this could be addressed with experimental evidence. At a minimum, this needs to be put in better context. There were a few concerns raised about the in vitro studies that need to be addressed. 1) Why are the levels of the CXCL16 and CX3CL1 similar in all cells where they are expressed, like the signal is on/off- type?

This question is indeed very interesting as this point might hint to a tissue/environment specific restriction of the “inverse signaling” and is therefore linked to the next reviewers’ comment. To address this question (expression of tm-chemokines = on/off?), we now show more ICC stainings for CXCL16 and CX3CL1 of selected cell lines in the bottom of Figure 1. These micrographs clearly indicate that expression levels are different in the respective (tumor) cell lines: while the brain tumor cell lines (glioma, neuroblastoma) show strong expression patterns, the breast cancer cell line MCF-7 shows only slight protein expression of the transmembrane chemokines. The LOX melanoma cell line does not express CXCL16 or CX3CL1 on protein level. Corresponding text passages have been added to the Results and the figure legend. Thus, protein expression corresponds to the mRNA expression levels obtained by qRT-PCR. However, these data are presented in a log-scale in Figure 1 (top). Thus, expression differences shown in the graph appear to be smaller than they are. To facilitate the readability of these data, we have added an explanation on ΔC_T values (a 3.33 alteration of ΔC_T value corresponds to a ten-fold altered expression) in this part of the Results section.

Together with the reviewers’ next comment on the cell specificity of the cellular responses this comment led us to the obvious question if the low but detectable expression level of MCF-7 cells affects the existence and dimension of the inverse signaling. Thus, we have performed some additional experiments addressing the activation of the MAP kinase pathway and the rescue from apoptosis also with MCF-7 cells, and added the respective results to the Supplementary material (Figure 3—figure supplement 2). Indeed, the tm-chemokine low expressing cell line MCF-7 shows only slight activation of MAP kinase signaling upon stimulation with s-chemokines, and the rescue from apoptosis is only mild, and hardly robust.

On this note, the data hint to a cell type specific expression strength rather than emphasizing an on/off signal. This is also supported by observations by us and others that both tm-chemokines are induced by pro-inflammatory cytokines, and therefore can be regulated by environmental conditions. A corresponding remark citing some examples in the literature has been added to the Discussion.

2) Why would all cell types respond with equal robust readouts? No cell-specific responses were provided, and I feel this to a bit odd.

This point is – as already explained above – related to the previous point as a specific response may on the one hand depend on the cell type or on the other hand on the respective expression level. Concerning different cell lines, we see different activation kinetics of the MAP kinase in different glioma cell lines upon stimulation with the s-chemokines (compare Figure 2). To, however, more properly address the raised concern, we performed stimulation experiments with a breast cancer cell line, MCF-7, expressing low levels of tm-chemokines. As mentioned above, this cell line showed also activation of the MAP kinase pathway and a rescue from apoptosis. However, these effects were clearly smaller and less robust in comparison to the tm-chemokine high expressing glioma cell lines. These data were added as supplementary material to Figure 3 (Figure 3—figure supplement 2), and a note was added to the Results.

Apart from this, in a separate manuscript, we show responses and effects of “inverse signaling” in meningioma cells that closely mirror the cellular effects of glioma cells as described in this manuscript. Briefly, primary human meningioma cells (meningioma: benign brain tumor descending from the meninges) showed activation of the MAP kinase pathway, enhanced proliferation and a reduction of caspase activity upon stimulation with CXCL16.

Taken together, our data emphasize an activation of the MAP kinase pathway and anti-apoptotic/proliferative effects of the “inverse signaling” of tm-chemokines that depends on the expression level of the tm-chemokines rather than the respective (tumor) cell type.

3) Figure 4C – In contrast to the stated conclusion, inhibition of CXCR7 appears to block the effects of the soluble chemokines (as there is not much more ERK phosphorylation compared with control when treated with inhibitor, compared with when the cells are treated with vehicle). I would advise repeating this assay to establish their conclusion with certainty.

This is a bit concerning because CXCR7 is a canonical receptor and its blockade should have no effect whatsoever since the whole premise of the paper is that canonical receptors are not required for inverse signaling. Thus it might be important to get them to sort this out.

To more substantiate this, we replaced the western blot in Figure 4C with a more convincing experiment, and added replications of this experiment to the supplementary figures (Figure 4—figure supplement 1). Additionally, to explain the phenomenon of non-canonical chemokine receptor signaling for a broader readership we have addressed this point in the Discussion.