Siglec1-expressing subcapsular sinus macrophages provide soil for melanoma lymph node metastasis
Lymphatic dissemination is a common metastasis route in solid cancers and LN metastasis is often associated with the severity of the disease ( Nathanson , 2003 ) . Although the clinical consequences of LN metastasis are debatable , recent reports have described clinically relevant events that take place during LN colonization by tumor cells ( Werner-Klein et al . , 2018; Pereira et al . , 2018; Brown et al . , 2018; Lee et al . , 2019; Naxerova et al . , 2017 ) . Melanoma metastasizes to LNs very early in cancer progression and metastatic cells acquire driver genetic changes during colonization of LNs , suggesting the important role that LNs have in tumor evolution ( Werner-Klein et al . , 2018 ) . These evolved post-LN-colonizing cancer cells with acquired colonization signatures show superior xenograft formation in mice compared with pre-colonizing cancer cells , suggesting a possible reason as to why metastasis-positive LNs are linked to disease severity in cancer ( Werner-Klein et al . , 2018 ) . Moreover , LNs provide a foothold for metastatic cells to disseminate further . Besides lymphatic dissemination to downstream LNs , during LN colonization metastatic cells gain access to the blood vessels in LNs and take a hematogenous route to colonize peripheral organs ( Pereira et al . , 2018; Brown et al . , 2018 ) . Despite all clinically important roles that LNs have in the systemic spread of evolved cancer cells , our knowledge of the early events of LN colonization by pioneer metastatic cells is very limited ( Naxerova et al . , 2017 ) . The distal organ metastatic colonization process is organ-specific and requires interactions between disseminated tumor cells ( DTCs; seed ) and distal organs ( soil ) to reinitiate growth ( Peinado et al . , 2017; Massagué and Obenauf , 2016 ) . Although primary tumors release many early DTCs into the circulation , which are the precursors of metastasis , very few eventually form distal metastasis . The majority of DTCs that land in unfamiliar distal sites fail to survive the hostile microenvironment of the new-found stroma . This inefficiency of metastasis suggests that distal organ colonization is a bottleneck of the metastasis colonization process . To gain a foothold in new landing sites , pioneer metastatic cells require active participation of the host stroma to obtain survival and viability signals ( Mehlen and Puisieux , 2006; Buchheit et al . , 2014 ) . A better understanding of the events that define this interaction between metastatic cells and host organ stroma is needed to identify actionable vulnerabilities of the metastatic colonization process . The current paradigm posits that LN metastatic colonization begins with the arrest of metastatic cells in the LN subcapsular sinus ( SCS ) ( Das et al . , 2013; Jeong et al . , 2015 ) . However , in LN metastasis , focus is often given to post-metastatic colonization events , whereas the interactions between metastatic cells and host stroma that leads to LN colonization by tumor cells remains largely uncharacterized ( Nathanson , 2003; Lee et al . , 2019; Das et al . , 2013; Jeong et al . , 2015; Olmeda et al . , 2017 ) . Therefore , using enhanced green fluorescent protein ( GFP ) -expressing mouse melanoma cells , we investigated the early LN metastatic colonization events and how LN supports the newly arrived DTCs .
To decipher the earliest metastatic events in LN , we first defined the stage at which melanoma LN metastasis formation takes place . Enhanced green fluorescent protein-expressing B16F10 ( B16-GFP ) melanoma cells were injected into mouse footpads to form primary tumor ( Figure 1—figure supplement 1A ) . The advantage of this model is that it mimics the natural course of metastasis , that is , primary tumor growth , pre-metastatic conditioning of draining LNs , and dissemination of tumor cells followed by LN colonization ( Jeong et al . , 2015; Harrell et al . , 2007 ) , while GFP enables us to track cancer cells even as single cells . Pioneer metastatic cells could be observed in the SCS of sentinel popliteal LNs lined by Lyve-1+ lymphatic endothelial cells as early as 2 weeks after inoculation and before the appearance of any visible macroscopic metastatic foci ( Figure 1—figure supplement 1B ) . Given the cell composition of the LN SCS and prominent presence of SCS macrophages in the sinus ( Gray and Cyster , 2012 ) , we reasoned that pioneer metastatic cells may use SCS macrophages to find an initial anchorage point . Therefore , we stained the LN sections positive for B16-GFP cells with anti-Siglec1 ( CD169 ) antibody , the surface protein highly expressed on macrophages on the floor of the SCS and in the LN medullary region ( Gray and Cyster , 2012; Nakamura et al . , 2002 ) . Of note , by depleting LN-resident macrophages through injecting clodronate liposomes into the mouse footpads , we confirmed that Siglec1 is primarily expressed on SCS and medullary sinus macrophages in LNs ( Figure 1—figure supplement 2 ) . We found the pioneer metastatic cells in close contact with SCS macrophages within the LN SCS , suggesting they interact with each other right after metastastic cells enter the SCS ( Figure 1A and B; Video 1 ) . Likewise , we confirmed this SCS macrophage–tumor cell interaction using another cell surface marker expressed on SCS macrophages , CD11b ( Figure 1—figure supplement 1C ) . We analyzed the sections from day 17 to day 21 , and found that after initial cancer cell attachment to SCS macrophages , small metastatic foci developed within the SCS ( Figure 1C ) . With the increase in size , the foci exerted pressure on the SCS macrophage–lymphatic lining , which eventually lead to disruption of the sinus lining and metastatic cells entering the LN cortex ( Figure 1D ) . Of note , to test the generalizability of our observations , we implanted an EGFP-expressing 4T1 mouse breast cancer cell line into mouse footpads and found that breast cancer cells also used SCS macrophages to find a foothold in LNs ( Figure 1—figure supplement 3 ) . Taken together with previous observations ( Das et al . , 2013; Jeong et al . , 2015 ) , where LN metastatic cells from different tumor types remain in the SCS and form a metastatic focus before invading into the LN cortex , our observations indicate that other tumors metastasizing to LNs may also use a LN colonization mechanism similar to melanoma . Increased protein sialylation is characteristic of several cancer types including melanomas ( Kim and Varki , 1997; Agrawal et al . , 2017 ) . Using Maackia amurensis II and Sambucus nigra lectins ( α−2 , 3- and α−2 , 6-sialylation-specific , respectively ) ( Varki , 2007 ) we found more than 10-fold higher cell surface α2 , 3‐ and α2 , 6‐linked sialylation in B16F10 melanoma cells compared with non-tumorigenic mouse melanocytes , melan-A cell line ( Figure 1—figure supplement 4A–D ) ( Bennett et al . , 1987 ) . Of note , melan-A cells showed only basal levels of α2 , 3-sialylation . As Siglec1 is a sialic-acid-recognizing protein that interacts with α2 , 3‐ and α2 , 6‐linked sialylated proteins ( Crocker et al . , 1999 ) , we hypothesized that it is Siglec1 itself that is responsible for the SCS macrophage–tumor cell interaction ( Nath et al . , 1999 ) . To confirm this , we used cell adhesion assay . HEK293T cells were grown as a monolayer in chamber slides and transfected with plasmid expressing mouse Siglec1 or empty plasmid as a mock control . We found significantly higher adherence of B16-GFP cells to Siglec1-expressing HEK293T cells compared with mock-transfected cells ( Figure 1E , F ) . Additionally , we confirmed Siglec1 binding to cancer cells by flow cytometry and microscopy employing recombinant mSiglec1 ( ECD ) -mFC protein ( Figure 1—figure supplement 4E , F ) . Furthermore , treatment with sialidase abolished the Siglec1 binding to cancer cells while removing the α2 , 3‐ sialylation completely and α2 , 6- sialylation by one third , suggesting Siglec1 preferentially binds to α2 , 3‐sialylated protein on cancer cells ( Figure 1—figure supplement 5A–F ) . Based on the above in vivo and in vitro results , we suggest that Siglec1-expressing SCS macrophages provide the ‘soil’ for incoming metastatic cells and prevent their washout with lymph flow . With limited chances of survival , disseminated tumor cells ( DTCs ) land in distal metastasis sites ( Massagué and Obenauf , 2016 ) . To establish a new colony , pioneer metastatic cells must overcome anoikis and amorphosis , and resume proliferation by establishing adhesive and signaling interactions with host tissue ( Mehlen and Puisieux , 2006; Buchheit et al . , 2014 ) . As our data confirmed cell-to-cell contact between SCS macrophages and pioneer metastatic cells , we investigated the functional consequences of this interaction for tumor cells . We used apoptosis marker cleaved-caspase-3 and cell proliferation marker Ki67 to determine the proliferation status of pioneer metastatic cells and found that they were in a non-proliferative non-apoptotic state when they landed in the LN SCS , and resumed proliferation after they came into contact with SCS macrophages ( Figure 2A–D ) . To confirm this , we used an in vitro co-culture method . Mouse Siglec1 was transiently expressed in HEK293T cells and B16-GFP cells were co-cultured with these Siglec1-expressing cells ( hereafter referred to as HEKSiglec1 and HEKMock for cells transfected with plasmid expressing mouse Siglec1 and empty plasmid , respectively; Figure 2E , Figure 2—figure supplement 1A ) . Of note , Siglec1 expression did not affect the proliferation status of HEK293T cells ( Figure 2—figure supplement 1B ) . To mimic the non-adherent nature of DTCs , we co-cultured cells in low binding culture plates . Of note , we observed that cancer cells cultured in non-adherent conditions showed significantly reduced Ki67 expression , similar to DTCs ( Figure 2—figure supplement 1C , D ) ( Pantel and Brakenhoff , 2004 ) . We found more Ki67+ proliferating B16-GFP cells in tumor cells-HEKSiglec1 co-cultures compared with cells co-cultured with HEKMock ( Figure 2F , G ) . To exclude the possibility that expression of Siglec1 may induce HEK293T cells to secrete some pro-proliferation factors and in turn induce B16-GFP cell proliferation , we prepared conditioned media from HEKMock and HEKSiglec1 cells and treated the B16-GFP cells in a similar setting as the co-culture experiments . Conditioned media did not affect the proliferation of B16-GFP cells ( Figure 2—figure supplement 1E , F ) . We next tested the role of Siglec1 in the survival of cancer cells . We assessed Annexin V staining in B16-GFP cells after co-culturing B16-GFP cells with HEKMock and HEKSiglec1 cells . B16-GFP cells co-cultured with HEKSiglec1 cells had significantly lower Annexin V-positive apoptotic cells ( Figure 2H , I ) . We further validated these results using a mouse macrophage cell line , J774A . 1 , which expresses Siglec1 ( Figure 2—figure supplement 2A ) . We used siRNA to knockdown Siglec1 expression in J774A . 1 cells and performed co-culture experiments with B16-GFP cells ( Figure 2—figure supplement 2B–D ) . We observed decreased proliferation of B16-GFP cells co-cultured with these Siglec1 knockdown J774A . 1 macrophages compared with B16-GFP cells co-cultured with control siRNA-transfected macrophages ( Figure 2—figure supplement 2E , F ) . Furthermore , we observed a significant increase in Annexin V-positive apoptotic cancer cells when they were co-cultured with Siglec1-knocked down macrophages ( Figure 2—figure supplement 2G , H ) . Taken together , our data revealed a role for Siglec1 in cancer cell survival and proliferation during the initial metastatic colonization of LNs by pioneer metastatic cells . Successful metastatic colonization requires activation of the pro-survival PI3K/AKT and ERK pathways ( Buchheit et al . , 2014 ) . Because these two cascades are crucial for metastatic colonization , we tested whether they were sensitive to Siglec1-dependent cancer cell interactions . After co-culturing B16-GFP cells with HEKSiglec1 or HEKMock for 18 hr , we performed intracellular Phosflow analysis . Siglec1 engagement with cancer cells induced AKT phosphorylation at S473 , which is a PI3K-mediated AKT-activating event ( Figure 2J , K ) . Furthermore , in a similar setting , we found higher levels of phosphorylated ERK1/2 ( ERK1 , T203/Y205; ERK2 , T183/Y185; Figure 2L , M ) in cancer cells co-cultured with HEKSiglec1 . We further analyzed AKT and ERK1/2 phosphorylation in B16-GFP cells co-cultured with Siglec1-knocked down J774A . 1 cells . Consistent with the reduced proliferation and increased apoptosis in B16-GFP co-cultured with Siglec1-knocked down J774A . 1 cells , we found reduced AKT and ERK1/2 phosphorylation in comparison with cells co-cultured with J774A . 1 cells transfected with control siRNA ( Figure 2—figure supplement 2I–L ) . Collectively , these results suggest that Siglec1 interactions provide growth cues to cancer cells . Therefore , these interactions may facilitate the LN colonization of melanoma cells . Next , to determine the molecular outcome of tumor cell and Siglec1 interactions , we performed a whole transcriptome analysis ( Figure 3A ) . Compared with tumor cells that were not interacting with Siglec1 , tumor cells cultured with HEKSiglec1 showed differential expression ( fold change >1 . 5; raw P<0 . 05 ) of 239 genes , of which 68 were upregulated and 171 were downregulated ( Figure 3B , Figure 3—source data 1 ) . A pathway analysis of the upregulated genes revealed that tumor cells from HEKSiglec1–tumor cell co-culture displayed enrichment in positive regulators of cell cycle and DNA replication compared with cancer cells cultured with mock-transfected HEK293T cells ( Figure 3C ) . Furthermore , gene ontology analysis revealed that upregulated genes were mainly involved in cell cycle-related biological processes ( Figure 3D , E ) . Consistent with FACS analysis of Ki67 staining , RNA sequencing results confirmed higher levels of Ki67 expression in HEKSiglec1 co-cultured cancer cells ( Figure 3E , Figure 3—source data 1 ) . Additionally , we observed downregulation of apoptosis-related ( Bbc3 , Rras , Ddit3 , Ddit4 ) and tumor suppressor ( Hes1 , Ahnak , Timp3 , Smad7 , Ndrg1 ) genes in Siglec1-interacting cancer cells ( Figure 3—source data 1 ) . Thus , the interaction between Siglec1 and cancer cells drove cell cycle progression and proliferation in cancer cells . In light of the prior knowledge of the vulnerabilities of metastatic processes , Siglec1-mediated interactions between SCS macrophages and metastatic cells may provide the vital support required to overcome dormancy and initiate successful LN colonization ( Pantel and Brakenhoff , 2004; Sosa et al . , 2014 ) . As we observed a shift towards increased cell cycle activity and proliferation in Siglec1-interacting cancer cells , we sought to further explain the mechanism by which Siglec1 interaction induces cell cycle activation in tumor cells by examining the critical mitotic events . To this end , we revisited our RNA sequencing results to identify important molecular events that regulate mitotic entry . Gene expression data showed significant upregulation in cell cycle master regulator PLK1 expression in co-culture experiments ( Figure 3E , Figure 3—source data 1 ) ( Paschal et al . , 2012 ) . This prompted us to compare PLK1 activation in primary tumor and early LN metastatic cells . We used Phosflow to compare PLK1 phosphorylation in primary tumor and LN metastatic cells . PLK1 phosphorylation at T210 is a mitosis-specific G2/M transition-activating event ( Paschal et al . , 2012 ) . We found 1 . 8-fold higher activation of PLK1 in LN metastatic cells in comparison with primary tumor cells ( Figure 4A , B ) . Consistently , in in vitro experiments , we found marked increase in mitosis-committed cancer cells when B16-GFP cells were co-cultured with HEKSiglec1 in comparison with HEKMock ( Figure 4C , D ) . With a reverse approach , where we knocked down Siglec1 using siRNA in J774A . 1 macrophages and performed co-culture experiments with B16-GFP cells , we found that knockdown of Siglec1 significantly decreased PLK1 phosphorylation ( Figure 4E–G , Figure 2—figure supplement 2A–D ) . Taken together , Siglec1-interacting tumor cells showed higher PLK1 phosphorylation than those that did not interact with Siglec1 . This strong mitotic induction observed in Siglec1-interacting tumor cells and confirmed by high PLK1 phosphorylation during early LN metastatic colonization supports our model whereby Siglec1 confers a growth advantage to pioneer metastatic cells . This presence of a highly growth permissive ‘soil’ reflects the high prevalence of LN metastasis in melanoma during the early stages of primary tumor growth , even before metastasis to peripheral organs . Hypersialylation in cancers , including melanomas , is directly linked to the metastatic potential of cancer cells ( Agrawal et al . , 2017; Bos et al . , 2009; Schultz et al . , 2012 ) . In previous experiments , we established that Siglec1 preferentially binds to α−2 , 3-linked sialylation ( Figure 1—figure supplement 5A–F ) . One sialyltransferase , ST3 beta-galactoside alpha-2 , 3-sialyltransferase 3 ( St3gal3 ) is known to produce Siglec-binding α−2 , 3 sialylation in mice and is linked to metastasis and poor prognosis in several cancer types ( Guo et al . , 2011; Pérez-Garay et al . , 2010; Cui et al . , 2011; Chang et al . , 2006 ) . Hence , we knocked out St3gal3 in B16-GFP cells using CRISPR-mediated single base pair deletion ( Figure 5—figure supplement 1A , B ) , which decreased α−2 , 3 sialylation of cell surface proteins by 20% ( Figure 5—figure supplement 1C , D ) . Additionally , St3gal3 knockout ( KO ) cells showed approximately 70% reduced cell surface binding of Siglec1 ( Figure 5—figure supplement 1E , F ) . Of note , St3gal3 KO did not affect the proliferation of B16-GFP cells in culture , in vitro adhesion and migration , or growth as primary tumors ( Figure 5—figure supplement 1G–K ) , but it abrogated the ability of B16-GFP to adhere to a Siglec1-expressing HEK293T cell monolayer ( Figure 5A , B ) . Notably , St3gal3 KO severely reduced the LN metastasis burden ( Figure 5C–E ) . To explore the possibility of Siglec1 expression on primary tumor lymphatic vessels , which might have affected the initial egress of cancer cells from the primary tumor , we searched for Siglec1 expression on primary tumor lymphatic vessels and found none ( Figure 5—figure supplement 2 ) . Next , we sought to explain the reduced metastasis by comparing the morphological features of early metastatic foci and quantifying the proliferation and survival of metastatic cells in the LN SCS . Immunohistological analysis at an early metastasis stage ( day 17 ) revealed that although St3gal3 KO B16-GFP cells are present in the LN SCS , they did not form foci and were not in contact with SCS macrophages ( Figure 5F ) . Whereas , WT B16-GFP cells formed tight-knit foci in contact with SCS macrophages ( Figure 5F ) . We further investigated whether this deprivation of Siglec1-derived signals from SCS macrophages affected the proliferation and survival of St3gal3 KO metastatic cells during early colonization . Ki67 staining revealed that St3gal3 KO B16-GFP cells showed severely reduced proliferation in the SCS in comparison with WT B16-GFP cells ( Figure 5G , H ) . Furthermore , early metastatic cells originating from St3gal3 KO cells showed significantly decreased survival in the SCS ( Figure 5I , J ) . Three weeks after tumor cell implantation , a large metastatic focus had formed in the LN , which disrupted the SCS macrophages lining the LN sinus , but St3gal3 KO tumors only formed small cell clusters , which crossed the SCS without growing in size in the SCS , suggesting a lack of ability to adhere to the SCS macrophages and hence reduced metastatic growth ( Figure 5—figure supplement 3 ) . Thus , overall α−2 , 3-sialylation abundance on tumor cells affects the efficiency of LN metastasis formation . Combined , these results suggest that the survival and proliferation of pioneer metastatic cells in the LN SCS is crucial process of metastatic colonization , and abrogation of SCS macrophage–tumor cell binding slowed down the metastatic colonization considerably .
Melanoma has a predilection for metastasizing to LNs in the early stages of the disease ( Bedrosian et al . , 2000 ) . The high prevalence of LN metastasis early during tumor progression suggests the presence of a metastasis-permissive environment in LNs . Our data provide a possible explanation for this . We propose a model whereby lymphatic disseminated metastatic cells directly interact with SCS macrophages just after landing in the LN SCS . The overall abundance and continuous presence of Siglec1+ macrophages in the SCS facilitates efficient metastatic seeding by providing adherence and promoting cell cycle progression in otherwise vulnerable DTCs . Our model is in agreement with the sequence of events leading to metastatic colonization of LN observed by Das et al . and Jeong et al . using in vivo imaging , whereby they observed that pioneer metastatic cells remain in the LN SCS to form metastatic foci that grow in size and disrupts the SCS to invade the LN cortex ( Das et al . , 2013; Jeong et al . , 2015 ) . LN SCS macrophages have been implicated in anti-tumor responses ( Asano et al . , 2011; Pucci et al . , 2016 ) . We show here another facet of Siglec1+ SCS macrophages where they physically interact with hypersialylated metastatic cells , conferring growth advantages upon them . Genes affected by Siglec1-cancer cell interactions were not only involved in cell cycle progression and DNA replication , but also in apoptosis and tumor suppression . Accordingly , reduced sialylation in tumor cells reduced the efficiency of metastasis . Our findings also suggest how the organ-specific LN metastasis trait of melanoma arises , and countering hypersialylation may therefore prove to be therapeutically beneficial in preventing LN metastasis and subsequent spread to distal organs .
Mouse melanoma cell line B16F10 ( ATCC ) and its derivatives , B16F10-GFP and St3gal3 knockout B16F10-GFP cell lines , were cultured in DMEM supplemented with 10% FBS and 2 mM l-glutamine . J774A . 1 ( ATCC ) mouse macrophage cells were cultured in DMEM supplemented with 10% FBS and 2 mM l-glutamine . 4T1 ( ATCC ) mouse breast cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS . HEK293T ( ATCC ) cells were cultured in DMEM medium supplemented with 10% FBS and 2 mM l-glutamine . Melan-A , a spontaneously immortalized melanocyte cell line , was kindly provided by Prof . Eun-Ju Chang , Asan Medical Center , Seoul , Korea . Melan-A cells were cultured in RPMI-1640 medium supplemented with 10% FBS , 200 nM phorbol 12-myristate 13-acetate ( PMA ) , 50 μg/mL streptomycin , and 50 U/mL penicillin . All cells tested negative for mycoplasma . pRGEN-CjCas9-CMV and pRGEN-U6-mSt3gal3-CjRG1 plasmids were purchased from Toolgen ( Seoul , South Korea ) with the following target sequence of RGEN: CAGTAAGTGTAGCTTCCAGGCAGAATAATAC ( underlined are PAM sequence-NNNNRYAC- not included in gRNA but recognized by Cas9 protein ) . B16F10-GFP cells were transfected with the abovementioned plasmids ( ratio 1:1 ) using Lipofectamine 3000 . Forty-eight hours after transfection , cells were single-cell sorted on a FACSAria cell sorter ( BD Biosciences , US ) and cultured in 96-well plates to isolate genomic DNA and PCR-amplify target regions . The following primers were used for amplifying target sites by Sanger DNA sequencing to select the KO cell line: forward primer 5′-TCACTATGCGGAGGAAGACTGCTTAATATC-3′ , reverse primer 5′-ATGCAGATTTCAAGGGTTGGGGGAAG-3′ . Forward primer was used for the DNA sequencing . C57/BL6 and BALB/c mice were purchased from OrientBio ( Gapyeong , Korea ) and were between 6 and 8 weeks of age at use . Mice were housed in specific pathogenfree conditions at the National Cancer Centre , Goyang . All experiments using animals were performed in accordance with protocols ( NCC-17–303C ) approved by the Institutional Animal Care and Use Committee of the National Cancer Centre and in accordance with the Guide for the Care and Use of Laboratory Animals . To study early LN metastatic cells , we implanted 2 × 105 B16F10-eGFP or 4T1-eGFP subcutaneously into one hind limb footpad of each anesthetized mouse ( Zoletil 40 mg/kg , Rompun 5 mg/kg ) . Animals were killed at different time points ( starting from day 10 ) to collect popliteal LNs . For LN metastasis assays , anesthetized mice were subcutaneously implanted with 2 × 105 B16F10-GFP or St3gal3 knockout B16F10-GFP cells in one hind limb footpad . Mice were killed at the end of the metastasis protocol , that is . , 21 days for the LN assessment . For total metastatic burden in LNs , LNs were processed as described under tissue dissociation for FACS staining sections . Primary tumor volumes were calculated using the following formula- volume ( mm3 ) = ( L × W2 ) /2 , where L ( length ) is the larger of two perpendicular axes and W ( width ) is the smaller of two perpendicular tumor axes . To confirm Siglec1 expression on the SCS macrophages , LN macrophages were depleted by footpad injection of 30 μL clodronate liposome . PBS liposomes were injected as control liposomes . Both liposomes were purchased from http://clodronateliposomes . org . Full-length mouse Siglec1 cDNA ( Transomic Technologies ) was cloned into mammalian expression plasmid pd18 . HEK293T cells were cultured in chamber slides to 70–80% confluency in DMEM supplemented with 10% FBS . At this point cells were transfected with empty vector ( pd18 ) or vector expressing mouse Siglec1 ( pd18-mSiglec1 ) using Lipofectamine 3000 ( Invitrogen ) . After 6 hr transfection , medium was changed , and cells were cultured for three days before use in cell adhesion assays . On day 3 , single cell suspensions of GFP-expressing cancer cells were prepared using enzyme-free PBS-based cell dissociation buffer ( Gibco ) and passing cells through 40 μm nylon filters ( Falcon ) . Cells were counted and 1 × 105 cells in 0 . 5 ml DMEM supplemented with 10% FBS were added per chamber . Cells were incubated for 5 min at 37°C , and then the medium was removed with non-attached cells and wells were further washed with complete medium three times with gentle force with a P1000 pipette . Cells were fixed by adding 2% paraformaldehyde directly into wells and incubating for 10 min at 37°C . Cells were stained as described in the immunostaining section . After image acquisition , adherent cells were counted using the Spots tool in Imaris ( version 8 . 1; Bitplane ) . For extracellular matrix cell adhesion assays , wells in 96-well plates were coated with 5 μg/ml fibronectin overnight at 4°C . The next day , wells were washed with PBS and then with DMEM . Single cell suspensions were prepared using enzyme-free cell dissociation buffer and cells were passed through 40 μm nylon filters . A total of 5 × 103 cells per well were added in a volume of 100 μl DMEM and incubated for 30 min at 37°C . After incubation , wells were washed with DMEM to remove unbound cells and 100 μl DMEM supplemented with 10% FBS was added to each well and incubated for 2 hr to allow cells to recover . Next , 20 μl CellTiter 96 AQueous One Solution ( Promega ) was added to each well and incubated for 1 hr at 37°C . After incubation , absorbance was measured at 490 nm using a 96-well plate reader . Fibronectin-coated wells with 100 μl DMEM supplemented with 10% FBS and without added cells were used to deduct the background . LNs were fixed with 4% paraformaldehyde and processed for cryosectioning . OCT-embedded tissues were sectioned by cryostat ( Leica ) in thin ( 20 μm ) or thick ( 100 μm ) sections . Thin sections were used to locate and visualize pioneer metastasis cells . All sections were inspected under the Zeiss Axio Imager Z1 microscope to locate sections with GFP-positive cells . For antibody staining , LN sections were permeabilized and blocked in 5% goat serum in PBST ( 0 . 3% Triton X-100 in PBS ) . Antibodies used for immunohistochemical staining are listed in the Supplementary Information . For staining in in vitro cell adhesion assays , cells in chamber slides were fixed with 2% paraformaldehyde and stained using a similar protocol as the LN staining . Sections were mounted in fluorescent mounting medium ( Dako ) and images were acquired on a LSM 780 and LSM 880 Laser Scanning Confocal Microscope ( Carl Zeiss SAS , Jena , Germany ) . All images were processed on Imaris ( version 8 . 1; Bitplane ) and shown as maximum intensity projections . Figure 3D reconstruction was performed on Imaris using the surface tool . HEK293T cells were grown in DMEM with 10% FBS , and 70–80% confluent cells were used for transfection with mammalian expression plasmids pd18 ( empty plasmid; mock ) or pd18-mSiglec1 using Lipofectamine 3000 ( Invitrogen ) . Medium was changed to CDM293 ( Invitrogen ) supplemented with L-glutamine and cells were further cultured for 3 days to ensure maximum mSiglec1 expression on the cell surface . On day 3 , single-cell preparations of B16-GFP cells were prepared by dissociating cells with enzyme-free PBS-based cell dissociation buffer ( Gibco ) and passing cells through 40 μm nylon filters ( Falcon ) . Both HEK293T cells and cancer cells were counted and mixed at a 4:1 ratio in DMEM supplemented with 5% FBS . Cells were cultured in low binding plates ( Corning ) for 18 hr at 37°C in 5% CO2 . At the end of the experiments , cells either underwent cell sorting to assess gene expression or were fixed in Cytofix buffer for Ki67 or Phosflow staining . For apoptosis quantification PE Annexin V ( BD biosciences ) was used according to manufacturer’s instructions . Pre-designed negative control and mouse Siglec1 siRNAs were obtained from Bioneer ( Bioneer , Daejeon , Korea ) . J774A . 1 cells were reverse-transfected with siRNA using Lipofectamine RNAiMAX transfection reagent ( Invitrogen ) in Opti-MEM medium . Medium was changed after 6 hr post-transfection to DMEM supplemented with 10% FBS . Siglec1 expression was assessed on day 3 and day four post-transfection using Alexa647-conjugated anti-mouse Siglec1 antibody . Co-culture experiments were performed as described in the previous section . For Ki67 and Phosflow ( pAKT , pERK , and pPLK1 ) analysis in co-culture experiments , cells were counted and fixed by adding Cytofix buffer directly into the cell culture , and cells were incubated for 10 min at 37°C . After washing with PBS , cells were permeabilized in 0 . 1% Triton X-100 in PBS for 5 min at room temperature . After washing the cells in staining buffer , the cells were incubated with mouse FcR blocking reagent ( Miltenyi Biotec ) . Then samples were incubated with Ki67 or Phosflow antibody in staining buffer . Following incubation , cells were washed and analyzed by FACSverse ( BD ) ; data were assessed using gates for GFP+ cells . Fold changes were calculated by dividing the mean fluorescence intensity of experimental samples by the mean fluorescence intensity of control samples . LNs and primary tumors were mechanically dissociated with forceps and scalpels and placed in DMEM containing 2 mg/ml collagenase ( Sigma ) for 20 min at 37°C ( for LN metastatic burden evaluation , collagenase incubation time was increased to 40 min ) . The suspensions were incubated in 0 . 1 mg/ml DNase I ( Roche ) for 10 mins . Tissue suspensions were resuspended with P1000 , filtered through 40 μm nylon filters ( Falcon ) , and aliquoted for total cell counting to adjust equal cell numbers for antibody staining . For antibody staining and FACS analysis , cells were fixed in 1 × Cytofix fixation buffer ( BD ) for 10 min at 37°C . After washing with PBS , cells were permeabilized in 0 . 1% Triton X-100 in PBS for 5 min at room temperature . Cells were washed and resuspended in staining buffer ( BD ) and then blocked with mouse FcR blocking reagent . Then samples were incubated with FACS antibodies and processed as described in the previous section . For lectin staining , 1 × 105 cells were resuspended in lectin staining buffer ( 10 mM HEPES , pH 7 . 5 , 0 . 15M NaCl , 0 . 09% sodium azide ) stained with 1 µg/ml lectin in a 100 µl volume at 4°C for 30 min followed by incubation with detection reagent streptavidin-PE for 20 min at 4°C . A total of 5 × 105 B16F10 cells were treated with 0 . 5 U/ml sialidase ( from Clostridium perfringens; Roche Applied Science ) in serum-free RPMI medium for 1 hr at 37°C in 5% CO2 and 95% relative humidity . Control cells were incubated in similar conditions but without the enzyme . After 1 hr , cells were washed in lectin staining buffer and stained with lectin or mouse Siglec1 ( ECD ) -mFc . Wild type and St3gal3 knockout B16F10-GFP cells were serum-starved overnight in DMEM medium . The next day , a single cell suspension was prepared using enzyme-free cell dissociation buffer and 5 × 104 cells were added in 100 μl to the upper chambers of 24-well transwell permeable supports with 8 μm pores ( Corning ) . The lower chambers were filled with 650 μl DMEM medium supplemented with 10% FBS as a chemoattractant . Control chambers were filled with DMEM without FBS . After 8 hr of incubation , the upper chambers were washed with PBS and non-migrated cells from the upper side of transwell chambers were removed gently using cotton swabs . Then cells on the lower side of the membranes were fixed with methanol for 10 min . Cells were stained with crystal violet solution for 10 min and thoroughly rinsed with water . After drying the membranes , images were captured on a Zeiss Axio Imager M1 ( Carl Zeiss ) microscope using a 10 × objective . The number of total migrated cells was counted using the following formula: ( average number on cells per image/image area ) × 0 . 33 cm2 . Next , the percentage of migrated cells was counted using the following formula: ( number of migrated cells/50 , 000 ) × 100 . To calculate the total number of GFP+ metastatic cells in LNs , the total number of LN cells was counted after tissue digestion . The number of metastatic cells was determined by gating around GFP+ cells and the following formula was used to calculate the final number of metastatic cells in LNs: ( number of cells in GFP+ gate/total number of FACS analyzed cells ) × total number of LN cells . After 18 hr of co-culture of GFP-expressing cancer cells with Siglec1-expressing or mock HEK293T cells in low binding plates , B16-GFP cells were sorted using a BD FACS Aria II or a BD FACS Aria SORP based on GFP expression . Total RNA from samples was isolated using a RNeasy Plus Mini kit ( Qiagen ) . Library preparation for RNA sequencing and data analysis was performed by Macrogen ( Macrogen Inc , Korea ) . Briefly , a TruSeq RNA library ( Illumine ) was constructed according to the manufacturer’s instructions and RNA-seq was performed on a HiSeq4000 ( Illumina ) . Processed trimmed reads were mapped to known reference genomes through the TopHat program ( version 2 . 0 . 13 ) and then aligned to UCSC mm10 using Bowtie aligner ( bowtie2 v2 . 2 . 3 ) , which allows splice junction processing . After read mapping , transcript assembly work was performed through the cufflinks program ( version 2 . 2 . 1 ) . As a result , the expression profile values for each sample were obtained for each transcript , and the FPKM ( Fragment per Kilobase of Transcript per Million mapped reads ) values were compiled based on the gene transcripts . Siglec1 and Dhfr were not included in the differential expressed gene counting and pathway analysis due to their plasmid origin . KEGG pathway analysis of unregulated genes was performed using the database for annotation , visualization and integrated discovery ( DAVID ) v6 . 8 ( Huang et al . , 2009a; Huang et al . , 2009b ) . GO enrichment analysis of unregulated genes was performed using the Gene Ontology ( GO ) knowledgebase ( Mi et al . , 2017 ) . All data are expressed as mean ±s . d . and data points are shown as scatter plots . Significance between two groups was analyzed with two-tailed , unpaired t-tests using Graphpad Prism software . Exact P-values are shown in the graphs .
Lymph nodes ( LNs ) are a common site of metastasis in solid cancers , and cutaneous melanomas show inherent properties of LN colonization . However , interactions between LN stroma and pioneer metastatic cells during metastatic colonization remain largely uncharacterized . Here we studied mice implanted with GFP-expressing melanoma cells to decipher early LN colonization events . We show that Siglec1-expressing subcapsular sinus ( SCS ) macrophages provide anchorage to pioneer metastatic cells . We performed in vitro co-culture to demonstrate that interactions between hypersialylated cancer cells and Siglec1 drive the proliferation of cancer cells . When comparing the transcriptome profile of Siglec1-interacting cancer cells against non-Siglec1-interacting cancer cells , we detected enrichment in positive regulators of cell cycle progression . Further , knockout of St3gal3 sialyltransferase compromised the metastatic efficiency of tumor cells by reducing α−2 , 3-linked sialylation . Thus , the interaction between Siglec1-expressing SCS macrophages and pioneer metastatic cells drives cell cycle progression and enables efficient metastatic colonization .
Cancer cells can leave the site where they arise and travel to other organs . Very few of the cancer cells that make this journey will survive long enough to form new tumors ( also known as ‘metastases’ ) . However , melanoma cells – the most aggressive type of skin cancer cells – are an exception . These cells will often colonize their nearest lymph nodes and melanoma patients with metastases in the lymph nodes are less likely to survive than those patients without them . Previous studies have shown that melanoma cells arrive at a lymph node and first proliferate in the region at the edge of this organ , known as the subcapsular sinus , before moving to the center . However , it was not understood how melanoma cells manage to survive in the subcapsular sinus . Now , Singh and Choi have tracked fluorescent melanoma cells to observe how they interact with the cells in the lymph nodes in mice . Melanoma cells have ‘sticky’ proteins coated with sugars on their surface . The results show that when the cells arrive in the subcapsular sinus these proteins bind to a receptor called Siglec1 located on the surface of immune cells called macrophages , which are also present . In this way , the melanoma cells anchor themselves in the lymph node . Moreover , binding Siglec1 helps melanoma cells survive and proliferate . In a last set of experiments , Singh and Choi deleted the enzyme responsible for making the sugar molecules in melanoma cells . Without the sugar coat , melanoma cells were less able to anchor themselves and grow within the mouse lymph nodes . Lymph nodes are often the first stop for melanoma cells on the way to other organs . Therefore , understanding the interaction between melanoma cells and macrophages might be useful for developing therapies that could disrupt this process and treat this aggressive cancer .