The `mxfda`

package contains tools for analyzing spatial
single-cell data, including multiplex imaging and spatial
transcriptomics, using methods from functional data analysis. Analyses
for this package are executed and stored using an S4 object of class
`mxFDA`

. This vignette outlines how to set up an
`mxFDA`

object from spatial single cell imaging data, how to
calculate spatial summary functions, and exploratory data analysis and
visualization of these spatial summary functions. Details on how to
perform downstream analysis and feature extraction using functional
principal component analysis can be found in the separate vignette
`vignette("mx_fpca")`

. To perform functional regression on
spatial summary functions from multiplex imaging data, see
`vignette("mx_funreg")`

.

Examples in this package use data adapted from the VectraPolarisData package on Bioconductor’s ExperimentHub. This package contains data from two multiplex imaging experiments conducted at the University of Colorado Anschutz Medical Campus. A shortcourse on single-cell multiplex imaging using these data is available here.

Data has been preprocessed and stored directly in the
`{mxfda}`

package. Available datasets are
`ovarian_FDA`

and `lung_df`

. This vignette will
focus on the `lung_df`

dataset, which contains a subset of 50
subjects from a multiplex imaging study of non-small cell lung carcinoma
described in Johnson et al. (2021). Each
subject has 3-5 multiplex images, which come from different regions of
interest (ROIs) in the tumor. Each ROI will be considered a “sample”
when constructing the `{mxfda}`

object. We load the lung
cancer data below.

The central object used with the `{mxfda}`

package is the
`mxFDA`

object. These objects are created with
`make_mxfda()`

and hold everything from the raw spatial data
to fit functional data models using derived spatial summary functions.
To save space on large samples, the metadata is kept separately from the
spatial data and when needed, is exported and merged together. Slots in
the `mxFDA`

object are designated as follows:

`Metadata`

- stores sample specific traits that may be used as covariates when fitting models`Spatial`

- a data frame of cell level information (x and y spatial coordinates, phenotype, etc.) that can be used to calculate spatial summary functions`subject_key`

- a character string for the column in the metadata that denotes the unique subject ID`sample_key`

- a character string for the column in the metadata that denotes the unique sample ID. Note that there may be multiple samples per subject, and this ID links the metadata and spatial data for each multiplex image sample`univariate_summaries`

and`bivariate_summaries`

- lists of spatial summary functions either imported with`add_summary_function()`

or calculated with`extract_summary_functions()`

`functional_pca`

- list of results from functional principle component analysis`functional_mpca`

- list of results from multilevel functional principle component analysis`functional_cox`

- list of functional cox models that have been fit

Using the `?lung_df`

in the `{mxfda}`

package,
columns with repeated data pertaining to the sample-level information
are extracted and stored in a data frame called `clinical`

while the cell-level information is kept in a long data frame
(`make_mxfda()`

also accepts cell-level information as a list
of data frames). The spatial parameter in the `make_mxfda()`

function can be left blank if using a spatial metric derived from
external functions. The final 2 parameters for the `?lung_df`

`mxFDA`

object are the `subject_key`

and the
`sample_key`

. The `sample_key`

is a column name
that appears both in the `metadata`

and `spatial`

and denotes unique samples while the `subject_key`

is a
column name in `metadata`

that ties the samples to metadata;
if the data contains one sample per subject then `sample_key`

and `subject_key`

is a 1:1, but if multiple samples per
subject, `subject_id`

will be repeated.

```
clinical = lung_df %>%
select(image_id, patient_id, patientImage_id, gender, age, survival_days, survival_status, stage) %>%
distinct()
spatial = lung_df %>%
select(-image_id, -gender, -age, -survival_days, -survival_status, -stage)
mxFDAobject = make_mxfda(metadata = clinical,
spatial = spatial,
subject_key = "patient_id",
sample_key = "patientImage_id")
```

Note that the object created has class `mxFDA`

.

The `{mxfda}`

package provides methods for analyzing
spatial relationships between cell types in single cell imaging data
based on point process theory. The location of cells in image samples
are treated as following a point process, realizations of a point
process are called “point patterns”, and point process models seek to
understand correlations in the spatial distributions of cells. Under the
assumption that the rate of a cell is constant over an entire region of
interest a point pattern will exhibit complete spatial randomness (CSR),
and it is often of interest to model whether cells deviate from CSR
either through clustering or repulsion. Spatial summary statistics can
be calculated to quantify the clustering and co-occurrence of cells in a
circular region with a particular radius *r*. Typically
univariate (one cell type) or bivariate (two cell types) summary
statistics are reported, and inference is obtained by comparing the
observed spatial summary statistic to that obtained under CSR. A popular
quantity is Ripley’s K(*r*), which studies the number of
neighbors to a particular point within radius *r*, and has
univariate and bivariate implementations in the `{spatstat}`

package (Baddeley, Rubak, and Turner
(2015)). Ripley’s K is characterized by clustering or repulsion
depending on whether it is above or below the theoretical value of \(\pi r^2\). Other spatial summary statistics
analyze the distance to a neighbor, and can be interpreted as
probabilities of observing a particular cell type within a radius
*r*. One of these metrics, G(*r*), or the nearest neighbor
distance distribution, is the cumulative distribution function of an
exponential random variable. More detailed overviews of spatial summary
functions for multiplex imaging data are provided in C. M. Wilson et al. (2021) and Wrobel, Harris, and Vandekar (2023).

Below we calculate univariate Ripley’s K to summarize the spatial
relationship among immune cells in each image. The `{mxfda}`

package accomplishes this with the function
`extract_summary_functions()`

. Either univariate or bivariate
can be calculated with this function depending on the choice supplied to
the `extract_func`

argument. To calculate a univariate
spatial summary we supply `univariate`

to the
`extract_func`

argument. The summary function that is
calculated depends on the function supplied to `summary_fun`

which is one of `Kest()`

, `Gest()`

, or
`Lest()`

from the `{spatstat.explore}`

package
(`Kcross()`

, `Gcross()`

, or `Lcross()`

for bivariate methods). Other options include supplying a vector of
radius values through `r_vec`

, and the a specific edge
correction (see Baddeley, Rubak, and Turner
(2015)). We calculate the K function across a range of radii from
0 to 100 and use the isotropic (“iso”) edge correction. See Baddeley, Rubak, and Turner (2015) for more
details on edge corrections for Ripley’s K and nearest neighbor G.

```
mxFDAobject = extract_summary_functions(mxFDAobject,
extract_func = univariate,
summary_func = Kest,
r_vec = seq(0, 100, by = 1),
edge_correction = "iso",
markvar = "immune",
mark1 = "immune")
```

Running this code will calculate univariate Ripley’s K function to
measure spatial clustering of immune cells for each sample, and will
store these spatial summary functions in the
`univariate_summaries`

slot of the `mxFDAobject`

.
To access this slot and view the extracted summary functions, type:

```
mxFDAobject@univariate_summaries$Kest
#> # A tibble: 24,847 × 6
#> patientImage_id r sumfun csr fundiff `immune cells`
#> <chr> <dbl> <dbl> <dbl> <dbl> <int>
#> 1 2_1 0 0 0 0 6
#> 2 2_1 1 0 3.14 -3.14 6
#> 3 2_1 2 0 12.6 -12.6 6
#> 4 2_1 3 0 28.3 -28.3 6
#> 5 2_1 4 0 50.3 -50.3 6
#> 6 2_1 5 0 78.5 -78.5 6
#> 7 2_1 6 0 113. -113. 6
#> 8 2_1 7 0 154. -154. 6
#> 9 2_1 8 0 201. -201. 6
#> 10 2_1 9 0 254. -254. 6
#> # ℹ 24,837 more rows
```

Note that the summaries are returned as a dataframe. The variable
`sumfun`

is the estimated summary function value,
`csr`

is the theoretical value under complete spatial
randomness, and `fundiff`

=
`sumfun`

-`csr`

describes the “degree of clustering
beyond what is expected due to chance; in downstream analysis we will
use the `fundiff`

covariate.

`mxFDA`

object`{mxfda}`

has S4 methods for visualization implemented via
the `plot()`

function (see `?plot.mxFDA`

for
details). The first argument is the `mxFDA`

object followed
by a few options that depend on what plot output is desired. Here, we
want to plot the univariate summary that we just calculated, which was
the K function. By passing in `what = 'uni k'`

, the plot
function will extract the univariate K results. We also need to tell
`plot()`

what column is the y-axis which can be
`'sumfun'`

for the observed value, `'csr'`

for the
theoretical value of complete spatial randomness (CSR), or
`'fundiff'`

which is the difference between the observed K
measure and the theoretical CSR. The output of `plot()`

is a
`{ggplot2}`

object which can then be easily added
to/manipulated as any ggplot plot would.

**NOTE: These are the columns when calculating using the
extract_summary_function() of {mxfda} but if
summary data is added from elsewhere with
add_summary_function() then those column names will have to
be used.**

The `extract_summary_functions()`

function can also be
used to extract bivariate summaries comparing spatial clustering of 2
cell types. We will look at relationship between T-cells and
macrophages. There are a few images that have fewer than 5 T-cells or
macrophages, which makes estimation of spatial summary functions less
stable for those images. To look at T-cells and macrophages, the data
phenotypes and cell locations have to be in long format so we first
create a variable with the cell types (`'phenotype'`

) from
the `lung_df`

.

```
lung_df = lung_df %>%
mutate(phenotype = case_when(phenotype_cd8 == "CD8+" ~ "T-cell",
phenotype_cd14 == "CD14+" ~ "macrophage",
TRUE ~ "other"),
phenotype = factor(phenotype))
```

We then recreate the mxFDAobject

```
spatial = lung_df %>%
select(-image_id, -gender, -age, -survival_days, -survival_status, -stage)
mxFDAobject = make_mxfda(metadata = clinical,
spatial = spatial,
subject_key = "patient_id",
sample_key = "patientImage_id")
```

Now we calculate the bivariate G function, but can replace
`Gcross()`

with `Lcross()`

or
`Kcross()`

to estimate the L or K bivariate functions
instead. The argument `markvar`

takes the variable that we
created above called `'phenotype'`

, and the 2 cell types that
we are interested in calculating the bivariate G for are
`'T-cell'`

and `'macrophage'`

so we provide them
to `mark1`

and `mark2`

, respectively.

```
mxFDAobject = extract_summary_functions(mxFDAobject,
summary_func = Gcross,
extract_func = bivariate,
r_vec = seq(0, 100, by = 1),
edge_correction = "rs",
markvar = "phenotype",
mark1 = "T-cell",
mark2 = "macrophage")
```

Just like with the univariate plots, we can use the
`plot()`

function to plot our `mxFDA`

object
results. The `what`

now is `'bi g'`

,
`'bivar g'`

, or `'bivariate g'`

.

Another useful function is `?summary.mxFDA`

which feeds
into the `summary()`

method. Either typing the name of the
object or wrapping it in the summary function will provide information
like the number of subjects, samples, if spatial summary functions have
been calculated, and functional data analyses that have been run.

```
mxFDAobject
#> mxFDA Object:
#> Subjects: 50
#> Samples: 247
#> Has spatial data
#> Univariate Summaries: None
#> Bivariate Summaries: Gcross
#> FPCs not yet calculated
#> MFPCs not yet calculated
#> FCMs not yet calculated
#> MFCMs not yet calculated
#> Scalar on Functional Regression not calculated
```

Sometimes other summary functions or normalizations are run outside
of the `{mxfda}`

package but the end goal is to still run
functional data analysis. Other packages, such as
`{spatialTIME}`

(Creed et al.
(2021)) provide methods for fast calculation of functions in
`{spatstat}`

with permutation estimates of complete spatial
randomness that are more robust than theoretical CSR estimates,
especially when tissue samples have holes that violate the assumption of
a homogeneous point pattern (see C. Wilson et al.
(2022)). Lets look at how to perform the estimation of univariate
nearest neighbor G with `{spatialTIME}`

.

The central object of `spatialTIME`

is the
`mIF`

object, that contains a list of spatial data frames, a
data frame of sample-level summaries, and a data frame for the metadata
(‘clinical’). From creating the `mxFDA`

object, we have a
spatial data frame and the clinical data, now we have to convert them
into something that works with `spatialTIME.`

The steps below
will be:

- convert positive/negative to 1/0 integers
- identify which columns in the spatial data frame are cell types
- convert spatial data frame to a list, where each element is a unique sample
- create a summary data frame from the spatial list for the number and proportion of positive cells for each phenotype

```
#Step 1
spatialTIME_spatial_df = spatial %>%
select(-phenotype) %>%
mutate(across(phenotype_ck:phenotype_cd4, ~ ifelse(grepl("\\+", .x), 1, 0))) %>%
relocate(patientImage_id, .before = 1)
#Step 2
cell_types = colnames(spatialTIME_spatial_df) %>% grep("phenotype", ., value = TRUE)
#Step 3
spatial_list = split(spatialTIME_spatial_df, spatial$patientImage_id)
#Step 4
summary_data = lapply(spatial_list, function(df){
df %>%
#group by sample ID to maintain ID column
group_by(patient_id, patientImage_id) %>%
#find number of positive
reframe(across(!!cell_types, ~ sum(.x)),
`Total Cells` = n()) %>%
#calculate proportion
mutate(across(!!cell_types, ~.x/`Total Cells` * 100, .names = "{.col} %"))
}) %>%
#bind the rows together
do.call(bind_rows, .)
```

With the spatial list, clinical, and summary data the
`mIF`

object can be constructed. For best computation
efficiency, use >v1.3.4.

```
library(spatialTIME)
#> Warning: package 'spatialTIME' was built under R version 4.3.2
#> spatialTIME version:
#> 1.3.4.3
#> If using for publication, please cite our manuscript:
#> https://doi.org/10.1093/bioinformatics/btab757
#make mif
mif = create_mif(clinical_data = clinical,
sample_data = summary_data,
spatial_list = spatial_list[1:50],
patient_id = "patient_id",
sample_id = "patientImage_id")
```

Deriving spatial metrics with the `mIF`

object is really
easy but does take some time. Will only do 10 permutations here to
estimate the complete spatial randomness measure of nearest neighbor G
and a reduced sampling, or `'rs'`

, edge correction. To make
the run faster, will look at only cytotoxic T cells (CD8+) and helper T
cells (CD4+).

```
mif = NN_G(mif, mnames = cell_types[c(2, 6)],
r_range = 0:100, num_permutations = 10,
edge_correction = "rs", keep_perm_dis = FALSE,
workers = 1, overwrite = TRUE, xloc = "x", yloc = "y")
```

With `spatialTIME`

, all cell types (markers) are added to
the data frame. We can visualize both CD8+ and CD4+ with ggplot.

```
mif$derived$univariate_NN %>%
ggplot() +
geom_line(aes(x = r, y = `Degree of Clustering Permutation`, color = patientImage_id), alpha = 0.4) +
facet_grid(~Marker) +
theme(legend.position = "none")
#> Warning: Removed 1086 rows containing missing values or values outside the scale range
#> (`geom_line()`).
```

Exporting the spatial summary function data from the `mIF`

object is the same as accessing the list object. However, we need to
make sure that the data that we use with the `mxFDA`

object
contains only a single cell types results. This is to make sure that
when modeling we aren’t mixing up different cells. Below is the
extraction and filtering of the new univariate G results and keeping
only the cytotoxic T cell results.

With the derived univariate nearest neighbor G for CD8+, it can be
added to an `mxFDA`

object with
`add_summary_function()`

. To show this, first will create a
new `mxFDA`

object with an empty spatial slot then add the
new summary function results.

```
#make mxFDA object
mxFDA_spatialTIME = make_mxfda(metadata = clinical,
spatial = NULL,
subject_key = "patient_id",
sample_key = "patientImage_id")
#add summary data
mxFDA_spatialTIME = add_summary_function(mxFDAobject,
summary_function_data = uni_g,
metric = "uni g")
```

Can now use the `mxfda`

plot method with the new data and
continue with analyses as would be done if using the internal
`extract_summary_function()`

.

Baddeley, Adrian, Ege Rubak, and Rolf Turner. 2015. *Spatial Point
Patterns: Methodology and Applications with r*. CRC press.

Creed, Jordan H, Christopher M Wilson, Alex C Soupir, Christelle M
Colin-Leitzinger, Gregory J Kimmel, Oscar E Ospina, Nicholas H
Chakiryan, et al. 2021. “spatialTIME and
iTIME: R package and Shiny application for visualization and analysis of
immunofluorescence data.” *Bioinformatics* 37 (23):
4584–86. https://doi.org/10.1093/bioinformatics/btab757.

Johnson, Amber M, Jennifer M Boland, Julia Wrobel, Emily K Klezcko, Mary
Weiser-Evans, Katharina Hopp, Lynn Heasley, et al. 2021. “Cancer
Cell-Specific Major Histocompatibility Complex II Expression as a
Determinant of the Immune Infiltrate Organization and Function in the
NSCLC Tumor Microenvironment.” *Journal of Thoracic
Oncology* 16 (10): 1694–704.

Wilson, Christopher M, Oscar E Ospina, Mary K Townsend, Jonathan Nguyen,
Carlos Moran Segura, Joellen M Schildkraut, Shelley S Tworoger, Lauren C
Peres, and Brooke L Fridley. 2021. “Challenges and Opportunities
in the Statistical Analysis of Multiplex Immunofluorescence
Data.” *Cancers* 13 (12): 3031.

Wilson, Christopher, Alex C Soupir, Ram Thapa, Jordan Creed, Jonathan
Nguyen, Carlos Moran Segura, Travis Gerke, Joellen M Schildkraut, Lauren
C Peres, and Brooke L Fridley. 2022. “Tumor Immune Cell Clustering
and Its Association with Survival in African American Women with Ovarian
Cancer.” *PLoS Computational Biology* 18 (3): e1009900.

Wrobel, Julia, Coleman Harris, and Simon Vandekar. 2023.
“Statistical Analysis of Multiplex Immunofluorescence and
Immunohistochemistry Imaging Data.” In *Statistical
Genomics*, 141–68. Springer.